TADG-15: an extracellular serine protease overexpressed in carcinomas

ABSTRACT

The present invention provides DNA encoding a TADG-15 protein as well as a TADG-15 protein. Also provided is a vector capable of expressing the DNA of the present invention adapted for expression in a recombinant cell and regulatory elements necessary for expression of the DNA in the cell. The present invention further provides for methods of inhibiting TADG-15 expression and/or protease activity methods of detecting TADG-15 mRNA and/or protein and methods of screening for TADG-15 inhibitors. Additionally, the present invention provides for cell-specific targeting via TADG-15 and methods of vaccinating an individual against TADG-15. The methods described are useful in the diagnosis. treatment and prevention of cancer particularly breast and ovarian cancer.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. Ser. No. 09/421,213filed on Oct. 20, 1999, which is a continuation-in-part of U.S. Ser. No.09/027,337, filed Feb. 20, 1998, now issued as U.S. Pat. No. 5,972,616.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of cellularbiology and the diagnosis of neoplastic disease. More specifically, thepresent invention relates to an extracellular serine protease, termedtumor antigen-derived gene 15 (TADG-15), which is overexpressed incarcinomas.

2. Description of the Related Art

Extracellular proteases have been directly associated with tumor growth,shedding of tumor cells and invasion of target organs. Individualclasses of proteases are involved in, but not limited to, (a) digestionof stroma surrounding the initial tumor area, (b) digestion of thecellular adhesion molecules to allow dissociation of tumor cells; and(c) invasion of the basement membrane for metastatic growth andactivation of both tumor growth factors and angiogenic factors.

In the process of cancer progression and invasion, proteases mediatespecific proteolysis and contribute to the removal of extracellularmatrix components surrounding tumor cells, the digestion ofintercellular adhesion molecules to allow dissociation of malignantcells and the activation of many growth and angiogenic factors.¹⁻³Depending on the nature of their catalytic domain, proteases areclassified into four families: serine proteases, metalloproteases,aspartic proteases and cysteine proteases.³ Among, these proteases, themetalloproteases have been well studied in relation to tumor growth andprogression, and they are known to be capable of degrading theextracellular matrix, thereby enhancing the invasive potential ofmalignant cells.^(1.4.5) For serine proteases, previous studies havedemonstrated an increased production of plasminogen activator in tumorcells and a positive correlation between plasminogen activator activityand aggressiveness of cancer.^(6.7) Prostate specific antigen (a serineprotease) has also been widely used as an indicator of abnormal prostategrowth.⁸ More recently, several other serine proteases have beenreported, viz. hepsin and the stratum corneum chymotryptic enzyme(SCCE), which are overexpressed in ovarian cancer and which maycontribute to malignant progression by increasing the extracellularlytic activity of these tumor cells.⁹

The prior art is deficient in the lack of effective means of screeningto identify proteases overexpressed in carcinoma. The present inventionfulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

The present invention discloses a screening program to identifyproteases overexpressed in carcinoma by examining PCR products amplifiedusing differential display in early stage tumors and metastatic tumorscompared to that of normal tissues. The approach herein to identifycandidate genes overexpressed in tumor cells has been to utilize thewell conserved domains surrounding the triad of amino acids(His-Asp-Ser) prototypical of the catalytic domain of serine proteases.Herein, evidence is presented for a unique form of serine protease notpreviously described in the literature which is highly expressed inovarian carcinomas. Through the screening approach using differentialPCR amplification of normal, low malignant potential and overtcarcinomas, a PCR product present only in carcinoma was subcloned andsequenced, and was found to have a catalytic domain which was consistentwith the serine protease family. Reported herein is the complete cloningand sequencing of this transcript and evidence for its expression inovarian tumor cells.

In one embodiment of the present invention, there is provided a DNAencoding a tumor antigen-derived gene (TADG-15) protein, selected fromthe following: (a) an isolated DNA which encodes a TADG-15 protein; (b)an isolated DNA which hybridizes under high stringency conditions to theisolated DNA of (a) above and which encodes a TADG-15 protein; and (c)an isolated DNA differing from the isolated DNAs of (a) and (b) above incodon sequence due to the degeneracy of the genetic code, and whichencodes a TADG-15 protein. The embodiment further includes a vectorcomprising the TADG-15 DNA and regulatory elements necessary forexpression of the DNA in a cell. Additionally embodied is a vector inwhich the TADG-15 DNA is positioned in reverse orientation relative tothe regulatory elements such that TADG-15 antisense mRNA is produced.

In another embodiment of the present invention, there is provided anisolated and purified TADG-15 protein coded for by DNA selected from thefollowing: (a) an isolated DNA which encodes a TADG-15 protein; (b) anisolated DNA which hybridizes under high stringency conditions toisolated DNA of (a) above and which encodes a TADG-15 protein; and (c)an isolated DNA differing from the isolated DNAs of (a) and (b) above incodon sequence due to the degeneracy of the genetic code, and whichencodes a TADG-15 protein.

In yet another embodiment of the present invention, there is provided amethod for detecting TADG-15 mRNA in a sample, comprising the steps of:(a) contacting a sample with a probe which is specific for TADG-15; and(b) detecting binding of the probe to TADG-15 mRNA in the sample. Instill yet another embodiment of the present invention, there is provideda kit for detecting TADG-15 mRNA, comprising: an oligonucleotide probespecific for TADG-15. A label for detection is further embodied in thekit.

The present invention additionally embodies a method of detectingTADG-15 protein in a sample, comprising the steps of: (a) contacting asample with an antibody which is specific for TADG-15 or a fragmentthereof; and (b) detecting binding of the antibody to TADG-15 protein inthe sample. Similarly, the present invention also embodies a kit fordetecting TADG-15 protein, comprising: an antibody specific for TADG-15protein or a fragment thereof. Means for detection of the antibody isfurther embodied in the kit.

In another embodiment, the present invention provides an antibodyspecific for the TADG-15 protein or a fragment thereof.

In yet another embodiment; the present invention provides a method ofscreening for compounds that inhibit TADG-15, comprising the steps of:(a) contacting a sample comprising TADG-15 protein with a compound; and(b) assaying for TADG-15 protease activity. Typically, a decrease in theTADG-15 protease activity in the presence of the compound relative toTADG-15 protease activity in the absence of the compound is indicativeof a compound that inhibits TADG-15.

In still yet another embodiment of the present invention, there isprovided a method of inhibiting expression of TADG-15 in a cell,comprising the step of: (a) introducing a vector into a cell, whereuponexpression of the vector produces TADG-15 antisense mRNA in the cellwhich hybridizes to endogenous TADG-15 mRNA, thereby inhibitingexpression of TADG-15 in the cell.

Further embodied by the present invention, there is provided a method ofinhibiting a TADG-15 protein in a cell, comprising the step of: (a)introducing an antibody specific for a TADG-15 protein or a fragmentthereof into a cell, whereupon binding of the antibody to the TADG-15protein inhibits the TADG-15 protein.

In an embodiment of the present invention, there is provided a method oftargeted therapy to an individual, comprising the step of: (a)administering a compound containing a targeting moiety and a therapeuticmoiety to an individual, wherein the targeting moiety is specific forTADG-15.

In an embodiment of the present invention, there is provided a method ofdiagnosing cancer in an individual, comprising the steps of: (a)obtaining a biological sample from an individual; and (b) detectingTADG-15 in the sample, wherein the presence of TADG-15 in the sample isindicative of the presence of carcinoma in the individual and theabsence of TADG-15 in the sample is indicative of the absence ofcarcinoma in the individual.

In another embodiment of the present invention, there is provided amethod of vaccinating an individual against TADG-15, comprising thesteps of: (a) inoculating an individual with a TADG-15 protein orfragment thereof that lacks TADG-15 protease activity, wherein theinoculation with the TADG-15 protein or fragment thereof elicits animmune response in the individual, thereby vaccinating the individualagainst TADG-15.

In an embodiment of the present invention, there is provided a method ofproducing immune-activated cells directed toward TADG-15, comprising thesteps of: exposing dendritic cells to a TADG-15 protein or fragmentthereof that lacks TADG-15 protease activity, wherein the exposure tosaid TADG-15 protein or fragment thereof activates the dendritic cells,thereby producing immune-activated cells directed toward TADG-15.

In another embodiment of the present invention, there is provided animmunogenic composition, comprising an immunogenic fragment of a TADG-15protein and an appropriate adjuvant.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention, as well as others which will become clear, areattained and can be understood in detail, more particular descriptionsof the invention briefly summarized above may be had by reference tocertain embodiments thereof which are illustrated in the appendeddrawings. These drawings form a part of the specification. It is to benoted, however, that the appended drawings illustrate preferredembodiments of the invention and therefore are not to be consideredlimiting in their scope.

The patent of application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A and 1B show a comparison of the serine protease catalyticdomain of TADG-15 with Hepsin (Heps, SEQ ID No. 3), SCCE (SEQ ID No. 4),Trypsin, (Try, SEQ ID No. 5), Chymotrypsin (Chymb, SEQ ID No. 6), Factor7 (Fac7, SEQ ID No. 7) and Tissue plasminogen activator (Tpa, SEQ ID No.8). The asterisks indicate conserved amino acids of catalytic triad.

FIGS. 2A-2D show the nucleotide sequence of the TADG-15 cDNA and thederived amino acid sequence of the TADG-15 protein. The putative startcodon is located at nucleotides 23-25. The potential transmembranesequence is underlined. Possible N-linked glycosylation sites areindicated by a broken line. The asterisks indicate conserved cysteineresidues of CUB domain. The SDE-motifs of the LDL receptor ligandbinding repeat-like domain are boxed. The arrow shows thearginine-valine bond cleaved upon activation. The conserved amino acidsof the catalytic triad; histidine, aspartic acid and serine residues arecircled.

FIG. 3 shows the amino acid sequence of the TADG-15 protease, includingfunctional sites and domains.

FIG. 4 shows a diagram of the TADG-15 protein. 1: cytoplasmic domain.(aa #1-54), 2; transmembrane domain (aa #55-57), 3; extracellular domain(aa #78-213). 4-5; CUB repeat (aa #214-447), 6-9; LDL receptor ligandbindings repeat (class A motif) like domain (aa #453-60), 10; serineprotease (aa #615-855).

FIG. 5 shows Northern blot analysis of TADG-15 mRNA expression in normalovary, ovarian carcinomas, carcinoma cell lines, normal fetal tissuesand normal adult tissues. A single intense transcript of the TADG-15 wasobserved in every sub-type of carcinoma and the two ovarian carcinomacell lines, SW626 and CAOV3, whereas no visible band was detected innormal ovary or the two breast cancer cell lines. In normal fetaltissues, fetal kidney showed increased transcript and faint expressionwas detected in fetal lung. In normal adult tissues, the TADG-15 wasdetected in colon with low expression in small intestine and prostate.

FIG. 6A shows quantitative PCR analysis of TADG-15 expression.Expression levels of TADG-15 relative to β-tubulin are significantlyelevated in all LMP tumors and carcinomas compared to that of normalovaries, m; mucinous, s; serous. FIG. 6B shows the ratio of TADG-15expression to expression of β-tubulin in normal ovary, LMP tumor andovarian carcinoma. TADG-15 mRNA expression levels were significantlyelevated in both LMP tumor (*; p<0.001) and carcinoma (**; p<0.0001)compared to that in normal ovary. All 10 samples of normal ovary showeda low level of TADG-15 expression.

FIG. 7 shows the TADG-15 expression in tumor cell lines derived fromboth ovarian and breast carcinoma tissues.

FIG. 8 shows the overexpression of TADG-15 in other tumor tissues.

FIG. 9 shows SW626 and CAOV3 cell lysates that were separated bySDS-PAGE and immunoblotted. Lanes 1 and 2 were probed with rabbitpre-immune serum as a negative control. Lanes 3 and 4 were probed withpolyclonal rabbit antibody generated to a carboxy terminal peptide fromTADG-15 protein sequence.

FIG. 10 shows that immunohistochemical staining of normal ovarianepithelium (FIG. 10A) with a polyclonal antibody to a TADG-15 proteasepeptide shows no staining of the stroma or epithelium. However, antibodystaining of carcinomas confirms the presence of TADG-15 expression inthe cytoplasm of a serous low malignant potential tumor (FIG. 10B); amucinous low malignant potential tumor (FIG. 10C): a serous carcinoma(FIG. 10D); and its presence in both the cytoplasm and cell surface of an endometrioid carcinoma (FIG. 10E).

FIGS. 11A and 11B show and alignment of the human TADG15 proteinsequence with that of mouse epithin which demonstrates that the proteinsare 84% similar and 81% identical over 843 amino acids. Residues thatare identical between the two proteins are indicated by a “-”, while he“*” symbol represents the TADG15 translation termination. The mostsignificant difference between these two proteins lies in thecarboxy-termini, which for epithin, includes 47 amino acids that are notpresent in TADG15.

FIGS. 12A-12E show a nucleotide sequence comparison between TADG-15 andhuman SNC-19 (GeneBank Accession No. #U20428).

DETAILED DESCRIPTION OF THE INVENTION

Proteases have been implicated in the extracellular modulation requiredfor tumor growth and invasion. In an effort to categorize thoseproteases contributing to ovarian carcinoma progression, redundantprimers directed towards conserved amino acid domains surrounding thecatalytic triad of His. Asp and Ser were utilized to amplify serineproteases differentially expressed in carcinomas. Using this method, aserine protease named TADG-15 (tumor antigen-derived gene 15) has beenidentified that is overexpressed in ovarian tumors. TADG-15 appears tobe a transmembrane multidomain serine protease. TADG-15 is highlyoverexpressed in ovarian tumors based on PCR, Northern blot andimmunolocalization.

The TADG-15 cDNA is 3147 base pairs long (SEQ ID No. 1) encoding for a855 amino acid protein (SEQ ID No. 2). The availability of the TADG-15gene provides numerous utilities. For example, the TADG-15 gene can beused as a diagnostic or therapeutic target in ovarian and othercarcinomas, including breast, prostate, lung and colon.

The present invention is directed to DNA encoding a tumorantigen-derived gene (TADG-15) protein, selected from the following: (a)an isolated DNA which encodes a TADG-15 protein; (b) an isolated DNAwhich hybridizes under high stringency conditions to the isolated DNA of(a) above and which encodes a TADG-15 protein; and (c) an isolated DNAdiffering from the isolated DNAs of (a) and (b) above in codon sequencedue to the degeneracy of the genetic code, and which encodes a TADG-15protein. It is preferred that the DNA has the sequence shown in SEQ IDNo. 1 and the TADG-15 protein has the amino acid sequence shown in SEQID No. 2.

The present invention is directed toward a vector comprising the TADG-15DNA and regulatory elements necessary for expression of the DNA in acell, or a vector in which the TADG-15 DNA is positioned in reverseorientation relative to the regulatory elements such that TADG-15antisense mRNA is produced. Generally, the DNA encodes a TADG-15 proteinhaving the amino acid sequence shown in SEQ ID No. 2. The invention isalso directed toward host cells transfected with either of theabove-described vector(s). Representative host cells are bacterialcells, mammalian cells, plant cells and insect cells. Preferably, thebacterial cell is E. Coli.

The present invention is directed toward an isolated and purifiedTADG-15 protein coded for by DNA selected from the following: (a) anisolated DNA which encodes a TADG-15 protein; (b) an isolated DNA whichhybridizes under high stringency conditions to isolated DNA of (a) aboveand which encodes a TADG-15 protein; and (c) an isolated DNA differingfrom the isolated DNAs of (a) and (b) above in codon sequence due to thedegeneracy of the genetic code, and which encodes a TADG-15 protein.Preferably, the protein has the amino acid sequence shown in SEQ ID No.2.

The present invention is directed toward a method for detecting TADG-15mRNA in a sample, comprising the steps of: (a) contacting a sample witha probe which is specific for TADG-15; and (b) detecting binding of theprobe to TADG-15 mRNA in the sample. The present invention is alsodirected toward a method of detecting TADG-15 protein in a sample,comprising the steps of: (a) contacting a sample with an antibody whichis specific for TADG-15 or a fragment thereof; and (b) detecting bindingof the antibody to TADG-15 protein in the sample. Generally, the sampleis a biological sample; preferably the biological sample is from anindividual; and typically, the individual is suspected of having cancer.

The present invention is directed toward a kit for detecting TADG-15mRNA, comprising: an oligonucleotide probe, wherein the probe isspecific for TADG-15. The kit may further comprise: a label with whichto label the probe; and means for detecting the label The presentinvention is additionally directed toward a kit for detecting TADG-15protein, comprising: an antibody which is specific for TADG-15 proteinor a fragment thereof. The kit may further comprise: means to detect theantibody.

The present invention is directed toward a antibody which is specificfor TADG-15 protein or a fragment thereof.

The present invention is directed toward a method of screening forcompounds that inhibit TADG-15, comprising the steps of: (a) contactinga sample containing TADG-15 protein with a compound; and (b) assayingfor TADG-15 protease activity. Typically, a decrease in the TADG-15protease activity in the presence of the compound relative to TADG-15protease activity in the absence of the compound is indicative of acompound that inhibits TADG-15.

The present invention is directed toward a method of inhibitingexpression of TADG-15 in a cell, comprising the step of: (a) introducinga vector expressing TADG-15 antisense mRNA into a cell, which hybridizesto endogenous TADG-15 mRNA, thereby inhibiting expression of TADG-15 inthe cell. Generally, the inhibition of TADG-15 expression is fortreating cancer.

The present invention is directed toward a method of inhibiting aTADG-15 protein in a cell, comprising the step of: (a) introducing anantibody specific for a TADG-15 protein or a fragment thereof into acell, which inhibits the TADG-15 protein. Generally, the inhibition ofthe TADG-15 protein is for treating cancer.

The present invention is directed toward a method of targeted therapy toan individual, comprising the step of: (a) administering a compoundhaving a targeting moiety and a therapeutic moiety to all individual,wherein the targeting moiety is specific for TADG-15. Representativetargeting moiety are a n antibody specific for TADG-15 and a ligand orligand binding domain (e.g., CUB, LDLR, protease and extracellular) thatbinds TADG-15. Likewise, a representative therapeutic moiety is aradioisotope, a toxin, a chemotherapeutic agent and immune stimulants.Typically, the above-described method is useful when the individualsuffers from ovarian cancer, breast cancer or cancers of the prostate,lung, colon and cervix.

The present invention is directed toward a method of diagnosing, cancerin an individual, comprising the steps of: (a) obtaining a biologicalsample from an individual; and (b) detecting TADG-15 in the sample.Generally, the presence of TADG-15 in the sample is indicative of thepresence of carcinoma in the individual, and the absence of TADG-15 inthe sample is indicative of the absence of carcinoma in the individual.Generally, the biological sample is blood, ascites fluid, urine, tears,saliva or interstitial fluid. Typical means of detecting TADG-15 are byNorthern blot. Western blot, PCR, dot blot, ELIZA, radioimmunoassay, DNAchips or tumor cell labelings. This method may be useful in diagnosingcancers such as ovarian, breast and other cancers in which TADG-15 isover-expressed, such as lung, prostate and colon cancers.

The present invention is also directed to an antisense oligonucleotidehaving the nucleotide sequence complementary to a TADG-15 mRNA sequence.The present invention is also directed to a composition comprising suchan antisense oligonucleotide according and a physiologically acceptablecarrier therefore.

The present invention is also directed to a method of treating aneoplastic state in an individual syndrome in an individual in need ofsuch treatment, comprising the step of administering to said individualan effective dose of an antisense oligonucleotide of. Preferably, theneoplastic state is selected from the group consisting of from ovariancancer, breast cancer, lung cancer, prostate cancer, colon cancer andother cancers in which TADG-15 is overexpressed. For such therapy, theoligonucleotides alone or in combination with other anti-neoplasticagents can be formulated for a variety of modes of administration,including systemic, topical or localized administration. Techniques andformulations generally can be found in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa. The oligonucleotide activeingredient is generally combined with a pharamceutically accceptablecarrier such as a diluent or excipient which can include fillers,extenders, binders, wetting agents, disintergrants, surface activeassents or lubricants, depending on the nature of the mode ofadministration and dosage forms. Typical dosage forms include tablets,powders, liquid preparations including suspensions, emulsions, andsolutions granules, capsules and suppositories, as well as liquidpreparations for injections, including liposome preparations.

For systemic administration, injection is preferred, includingintramuscular, intravenous, intraperitoneal and subcutaneous. Forinjection, the oligonucleotides of the invention are formulated inliquid solutions, preferably in physiologically compatible buffers. Inaddition, the oligonucleotides can b e formulated in solid form andredissolved or suspended immediately prior to use. Lyophilized forms arealso included. Dosages that can be used for systemic administrationpreferably range from about 0.01 mg/kg to 50 mg/kg administered once ortwice per day. However, different dosing schedules can be utilizeddepending on (1) the potency of an individual oligonucleotide atinhibiting the activity of its target DNA, (2) the severity or extent ofthe pathological disease state, or (3) the pharmacokinetic behavior of agiven oligonucleotide.

The present invention is directed toward a method of vaccinating andindividual against TADG-15 overexpression, comprising the steps of: (a)inoculating an individual with a TADG-15 protein or fragment thereofwhich lacks TADG-15 protease activity. The inoculation with the TADG-15protein or fragment thereof elicits an immune response in theindividual, thereby vaccinating the individual against TADG-15. Thevaccination with TADG-15 described herein is intended for an individualwho has cancer, is suspected of having cancer or is at risk of gettingcancer. Generally, the TADG-15 fragment useful for vaccinating anindividual are 9-residue fragments up to 20-residue fragments, withpreferred 9-residue fragments shown in SEQ ID Nos. 2, 19, 20, 21, 29,39, 49, 50, 59, 79, 80, 81, 82, 83, 84, 89 and 90.

The present invention is directed toward a method of producingimmune-activated cells directed toward TADG-15, comprising the steps of:exposing dendritic cells to a TADG-15 protein or fragment thereof thatlacks TADG-15 protease activity, wherein exposure to the TADG-15 proteinor fragment thereof activates the dendritic cells, thereby producingimmune-activated cells directed toward TADG-15. Representativeimmune-activated cells are B-cells, T-cells and dendrites. Generally,the TADG-15 fragment is a 9 -residue fragment up to a 20-residuefragment, with preferable 9 -residue fragments shown in SEQ ID Nos. 2,19, 20, 21, 29, 39, 49, 50, 59, 79, 80, 81, 82, 83, 84, 89 and 90.Preferably, the dendritic cells are isolated from an individual prior toexposure, and the activated dendritic cells reintroduced into theindividual subsequent to exposure. Typically, the individual for whichthis method may apply has cancer, is suspected of having cancer or is atrisk of getting cancer.

The present invention is directed toward an immunogenic composition,comprising an immunogenic fragment of a TADG-15 protein and anappropriate adjuvant. Generally, the fragment is a 9-residue fragment upto a 20-residue fragment, with preferred 9-residue fragments shown inSEQ ID Nos. 2, 19, 20. 21, 29, 39, 49, 50, 59, 79, 80, 81, 82, 83, 84,89 and 90.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques arc explainedfully in the literature. See, e.g., Maniatis, Fritsch & Sambrook.“Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning,: APractical Approach,” Volumes I and II (D. N. Glover ed. 1985);“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” (B. D. Hames & S. J. Higgins eds. 1985); “Transcriptionand Translation” (B. D. Hames & S. J. Higgins eds. 1984); “Animal CellCulture” (R. I. Freshney, ed. 1986); “Immobilized Cells And Enzymes”(IRL Press, 1986); B. Perbal, “A Practical Guide To Molecular Cloning”(1984). Therefore, if appearing herein, the following terms shall havethe definitions set out below.

As used herein, the term “cDNA” shall refer to the DNA copy of the mRNAtranscript of a gene.

As used herein, the term “derived amino acid sequence” shall mean theamino acid sequence determined by reading the triplet sequence ofnucleotide bases in the cDNA.

As used herein the term “screening a library” shall refer to the processof using a labeled probe to check whether, under the appropriateconditions, there is a sequence complementary to the probe present in aparticular DNA library. In addition, “screening a library” could beperformed by PCR.

As used herein, the term “PCR” refers to the polymerase chain reactionthat is the subject of U.S. Pat. Nos. 4,683,195 and 4,683,202 to Mullis,as well as other improvements now known in the art.

The amino acid described herein are preferred to be in the “L” isomericform. However, residues in the “D” isomeric form can be substituted forany L-amino acid residue, as long as the desired functional property ofimmunoglobulin-binding is retained by the polypeptide. NH₂ refers to thefree amino group present at the amino terminus of a polypeptide. COOHrefers to the free carboxy group present at the carboxy terminus of apolypeptide. In keeping with standard polypeptide nomenclature, J Biol.Chem., 243:3552-59 (1969), abbreviations for amino acid residues areused as in customary in the art.

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid residues.

A “replicon” is any genetic element (e.g., plasmid, chromosome, virus)that functions as an autonomous unit of DNA replication in vivo; i.e.,capable of replication under its own control.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. A “vector” may further be defined as areplicable nucleic acid construct, e.g., a plasmid or viral nucleicacid.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single-strandedform or as a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. The structure isdiscussed herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

An expression vector is a replicable construct in which a nucleic acidsequence encodings a polypeptide is operably linked to suitable controlsequences capable of effecting expression of the polypeptide in a cell.The need for such control sequences will vary depending upon the cellselected and the transformation method chosen. Generally, controlsequences include a transcriptional promoter and/or enhancer, suitablemRNA ribosomal binding sites, and sequences which control thetermination of transcription and translation. Methods which are wellknown to those skilled in the art can be used to construct expressionvectors containing appropriate transcriptional and translational controlsignals. See, for example, techniques described in Sambrook et al.,1989, Molecular Cloning: A Laboratory Manual (2nd Ed.), Cold SpringHarbor Press, N.Y. A gene and its transcription control sequences aredefined as being “operably linked” if the transcription controlsequences effectively control transcription of the gene. Vectors of theinvention include, but are not limited to plasmid vectors and viralvectors. Preferred viral vectors of the invention are those derived fromretroviruses, adenovirus, adeno-associated virus, SV40 virus, or herpesviruses. In general, expression vectors contain promoter sequences whichfacilitate the efficient transcription of the inserted DNA fragment andare used in connection with the host. The expression vector typicallycontains an origin of replication, promoter(s), terminator(s), as wellas specific genes which are capable of providing phenotypic selection intransformed cells. The transformed hosts can be fermented and culturedaccording to means known in the art to achieve optimal cell growth.

An “origin of replication” refers to those DNA sequences thatparticipate in DNA synthesis.

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, polyadenylation signals,terminators, and the like, that provide for the expression of a codingsequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site, as well asprotein binding domains (consensus sequences) responsible for thebinding of RNA polymerase. Eukaryotic promoters often, but not always,contain “TATA” boxes and “CAT” boxes. Prokaryotic promoters typicallycontain Shine-Dalgarno ribosome-binding sequences in addition to the −10and −35 consensus sequences.

An “expression control sequence” is a DNA sequence that controls andregulates the transcription and translation of another DNA sequence. Acoding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then translated intothe protein encoded by the coding sequence.

A “signal sequence” can be included near the coding sequence. Thissequence encodes a signal peptide. N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to enzymes, each of which cut double-stranded DNA at ornear a specific nucleotide sequence.

A cell has been “transformed” by exogenous or heterologous DNA when suchDNA has been introduced inside the cell. The transforming DNA may or maynot be integrated (covalently linked) into the genome of the cell. Inprokaryotes, yeast, and mammalian cells for example, the transformingDNA may be maintained on an episomal element such as a plasmid. Withrespect to eukaryotic cells, a stably transformed cell is one in whichthe transforming DNA has become integrated into a chromosome so that itis inherited by daughter cells through chromosome replication. Thisstability is demonstrated by the ability of the eukaryotic cell toestablish cell lines or clones comprised of a population of daughtercells containing the transforming DNA. A “clone” is a population ofcells derived from a single cell or ancestor by mitosis. A “cell line”is a clone of a primary cell that is capable of stable growth vitro formany generations.

Two DNA sequences are “substantially homologous” when at least about 75%(preferably at least about 80%, and most preferably at least about 90%or 95%) of the nucleotides match over the defined length of the DNAsequences. Sequences that are substantially homologous can be identifiedby comparing the sequences using standard software available in sequencedata banks, or in a Southern hybridization experiment under, forexample, stringent conditions as defined for that particular system.Defining appropriate hybridization conditions is within the skill of theart. See, e.g., Maniatis et al., supra; DNA Cloning, Vols. I & II,supra; Nucleic Acid Hybridization, supra.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. In another example, coding sequence is a construct where thecoding sequence itself is not found in nature (e.g., a cDNA where thegenomic coding sequence contains introns, or synthetic sequences halvingcodons different than the native gene). Allelic variations ornaturally-occurring mutational events do not give rise to a heterologousregion of DNA as defined herein.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others. A number of fluorescent materials are known and canbe utilized as labels. These include, for example, fluorescein,rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. Aparticular detecting material is anti-rabbit antibody prepared in goatsand conjugated with fluorescein through an isothiocyanate. Proteins canalso be labeled with a radioactive element or with an enzyme. Theradioactive label can be detected by any of the currently availablecounting procedures. The preferred isotope may be selected from ³H, ¹⁴C,³²P, ³⁵S, ³⁶Cl ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³¹I, and ¹⁸⁶Re.Enzyme labels are likewise useful, and can be detected by any of thepresently utilized colorimetric, spectrophotometric,fluorospectrophotometric, amperometric or gasometric techniques. Theenzyme is conjugated to the selected particle by reaction with bridgingmolecules such as carbodiimides. diisocyanates, glutaraldehyde and thelike. Many enzymes which can be used in these procedures are known andcan be utilized. The preferred are peroxidase. β-glucuronidase,β-D-glucosidase, β-D-galactosidase, urease, glucose oxidase plusperoxidase and alkaline phosphatase. U.S. Pat. Nos. 3,654,090,3,850,752, and 4,016,043 are referred to by way of example for theirdisclosure of alternate labeling material and methods.

A particular assay system developed and utilized in the art is known asa receptor assay. In a receptor assay, the material to be assayed isappropriately labeled and then certain cellular test colonies areinoculated with a quantitiy of both the label after which bindingstudies are conducted to determine the extent to which the labeledmaterial binds to the cell receptors. In this way, differences inaffinity between materials can be ascertained.

An assay useful in the art is known as a “cis/trans” assay. Briefly,this assay employs two genetic constructs, one of which is typically aplasmid that continually expresses a particular receptor of interestwhen transfected into an appropriate cell line, and the second of whichis a plasmid that expresses a reporter such as luciferase, under thecontrol of a receptor/ligand complex. Thus, for example, if it isdesired to evaluate a compound as a ligand for a particular receptor,one of the plasmids would be a construct that results in expression ofthe receptor in the chosen cell line, while the second plasmid wouldpossess a promoter linked to the luciferase gene in which the responseelement to the particular receptor is inserted. If the compound undertest is an agonist for the receptor, the ligand will complex with thereceptor, and the resulting complex will bind the response element andinitiate transcription of the luciferase gene. The resultingchemiluminescence is then measured photometrically, and dose responsecurves are obtained and compared to those of known ligands. Theforegoing protocol is described in detail in U.S. Pat. No. 4,981,784.

As used herein, the term “host” is meant to include not only prokaryotesbut also eukaryotes such as yeast, plant and animal cells. A recombinantDNA molecule or gene which encodes a human TADG-15 protein of thepresent invention can be used to transform a host using any of thetechniques commonly known to those of ordinary skill in the art.Especially preferred is the use of a vector containing coding sequencesfor the gene which encodes a human TADG-15 protein of the presentinvention for purposes of prokaryote transformation. Prokaryotic hostsmay include E. coli. S. tymphimurium, Serratia marcescens and Bacillussubtilis. Eukaryotic hosts include yeasts such as Pichia pastoris,mammalian cells and insect cells.

The invention includes a substantially pure DNA encoding a TADG-15protein, a DNA strand which will hybridize at high stringency to a probecontaining a sequence of at least 15 consecutive nucleotides of (SEQ IDNo. 1). The protein encoded by the DNA of this invention may share atleast 80% sequence identity (preferably 85%, more preferably 90%, andmost preferably 95%) with the amino acids listed in FIGS. 3 and 4 (SEQID No. 2). More preferably, the DNA includes the coding sequence of thenucleotides of FIGS. 2A-2D (SEQ ID No. 1), or a degenerate variant ofsuch a sequence. This invention also includes a substantially pure DNAcontaining a sequence of at least 15 consecutive nucleotides (preferably20, more preferably 30, even more preferably 50, and most preferablyall) of the region from nucleotides 1 to 3147 of the nucleotides shownin FIGS. 2A-2D (SEQ ID No. 1).

By “substantially pure DNA” is meant DNA that is not part of a milieu inwhich the DNA naturally occurs, by virtue of separation (partial ortotal purification) of some or all of the molecules of that milieu, orby virtue of alteration of sequences that flank the claimed DNA. Theterm therefore includes, for example, a recombinant DNA which isincorporated into a vector, into an autonomously replicating plasmid orvirus, or into the genomic DNA of a prokaryote or eukaryote; or whichexists as a separate molecule (e.g., a cDNA or a genomic or cDNAfragment produced by polymerase chain reaction (PCR) or restrictionendonuclease digestion) independent of other sequences. It also includesa recombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence, e.g., a fusion protein. Also included is arecombinant DNA which includes a portion of the nucleotides listed inFIGS. 2A-2D (SEQ ID No. 1) and which encodes an alternative splicevariant of TADG-15.

By a “substantially pure protein” is meant a protein which has beenseparated from at least some of those components which naturallyaccompany it. Typically, the protein is substantially pure when it is atleast 60% (by weight) free from the proteins and othernaturally-occurring organic molecules with which it is naturallyassociated in vivo. Preferably, the purity of the preparation (byweight) is at least 75%, more preferably at least 90%, and mostpreferably at least 99%. A substantially pure TADG-15 protein may beobtained, for example, by extraction from a natural source; byexpression of a recombinant nucleic acid encoding a TADG-15 polypeptide;or by chemically synthesizing the protein. Purity can be measured by anyappropriate method, e.g., column chromatography, such as immunoaffinitychromatography using an antibody specific for TADG-15, polyacrylamidegel electrophoresis, or HPLC analysis. A protein is substantially freeof naturally associated components when it is separated from at leastsome of those contaminants which accompany it in its natural state.Thus, a protein which is chemically synthesized or produced in acellular system different from the cell from which it naturallyoriginates will be, by definition, substantially free from its naturallyassociated components. Accordingly, substantially pure proteins includeeukaryotic proteins synthesized in E. Coli, other prokaryotes, or anyother organism in which they do not naturally occur.

The term “oligonucleotide”, as used herein, is defined as a moleculecomprised of two or more ribonucleotides, preferably more than three.Its exact size will depend upon many factors, which, in turn, dependupon the ultimate function and use of the oligonucleotide. The term“primer”, as used herein, refers to an oligonucleotide, whetheroccurring naturally (as in a purified restriction digest) or producedsynthetically, and which is capable of initiating synthesis of a strandcomplementary to a nucleic acid when placed under appropriateconditions, i.e., in the presence of nucleotides and an inducing agent,such as a DNA polymerase, and at a suitable temperature and pH. Theprimer may be either single-stranded or double-stranded and must besufficiently long to prime the synthesis of the desired extensionproduct in the presence of the inducing agent. The exact length of theprimer will depend upon many factors, including temperature, sequenceand/or homology of primer and the method used. For example, indiagnostic applications, the oligonucleotide primer typically contains15-25 or more nucleotides, depending upon the complexity of the targetsequence, although it may contain fewer nucleotides.

The primers herein are selected to be “substantially” complementary toparticular target DNA sequences. This means that the primers must besufficiently complementary to hybridize with their respective strands.Therefore, the primer sequence need not reflect the exact sequence ofthe template. For example, a non-complementary nucleotide fragment(i.e., containing a restriction site) may be attached to the 5′ end ofthe primer, with the remainder of the primer sequence beingcomplementary to the strand. Alternatively, non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer sequence has sufficient complementary with the sequence tohybridize therewith and form the template for synthesis of the extensionproduct.

The probe to which the DNA of the invention hybridizes preferablyconsists of a sequence of at least 20 consecutive nucleotides, morepreferably 40 nucleotides, even more preferably 50 nucleotides, and mostpreferably 100 nucleotides or more (up to 100%) of the coding sequenceof the nucleotides listed in FIGS. 2A-2D (SEQ ID No. 1) or thecomplement thereof. Such a probe is useful for detecting expression ofTADG-15 in a cell by a method including the steps of (a) contacting mRNAobtained from the cell with a labeled TADG-15 hybridization probe; and(b) detecting hybridization of the probe with the mRNA.

By “high stringency” is meant DNA hybridization and wash conditionscharacterized by high temperature and low salt concentration, e.g., washconditions of 65° C. at a salt concentration of approximately 0.1×SSC,or the functional equivalent thereof. For example, high stringencyconditions may include hybridization at about 42° C. in the presence ofabout 50% formamide; a first wash at about 65° C. with about 2×SSCcontaining 1% SDS; followed by a second wash at about 65° C. with about0.1×SSC.

The DNA may have at least about 70% sequence identity to the codingsequence of the nucleotides listed in FIGS. 2A-2D (SEQ ID No. 1),preferably at least 75% (e.g., at least 80%); and most preferably atleast 90%. The identity between two sequences is a direct function ofthe number of matching or identical positions. When a position in bothof the two sequences is occupied by same monomeric subunit e.g., if agiven position is occupied by an adenine in each of two DNA molecules,then they are identical at that position. For example, if 7 positions ina sequence 10 nucleotides in length are identical to the correspondingpositions in a second 10-nucleotide sequence, then the two sequenceshave 70% sequence identity. The length of comparison sequences willgenerally be at least 50 nucleotides, preferably at least 60nucleotides, more preferably at least 75 nucleotides, and mostpreferably 100 nucleotides. Sequence identity is typically measuredusing sequence analysis software (e.g., Sequence Analysis SoftwarePackage of the Genetics Computer Group (GCG), University of WisconsinBiotechnology Center, 1710 University Avenue, Madison, Wis. 53705).

The present invention comprises a vector comprising a DNA sequence whichencodes a human TADG-15 protein, wherein said vector is capable ofreplication in a host, and comprises, in operable linkage: a) an originof replication; b) a promoter: and c) a DNA sequence coding for saidTADG-15 protein. Preferably, the vector of the present inventioncontains a portion of the DNA sequence shown in SEQ ID No. 1. Vectorsmay be used to amplify and/or express nucleic acid encoding a TADG-15protein or fragment thereof.

In addition to substantially full-length proteins, the invention alsoincludes fragments (e.g., antigenic fragments) of the TADG-15 protein(SEQ ID No. 2). As used herein, “fragment,” as applied to a polypeptide,will ordinarily be at least 6 residues, more typically at least 9-12residues, and preferably at least 13-20 residues in length, but lessthan the entire, intact sequence. Alternatively, a fragment may be anindividual domain of 20-120 residues from SEQ ID No. 2. Fragments of theTADG-15 protein can be generated by methods known to those skilled inthe art, e.g., by enzymatic digestion of naturally occurring orrecombinant TADG-15 protein, by recombinant DNA techniques using anexpression vector that encodes a defined fragment of TADG-15, or bychemical synthesis. The ability of a candidate fragment to exhibit acharacteristic of TADG-15 (e.g., binding to an antibody specific forTADG-15) can be assessed by methods described herein. Purified TADG-15or antigenic fragments of TADG-15 can be used to generate new antibodiesor to test existing antibodies (e.g., as positive controls in adiagnostic assay) by employing standard protocols known to those skilledin the art. Included in this invention is polyclonal antisera generatedby using TADG-15 or a fragment of TADG-15 as the immunogen in, e.g.,rabbits. Standard protocols for monoclonal and polyclonal antibodyproduction known to those skilled in this art are employed. Themonoclonal antibodies generated by this procedure can be screened forthe ability to identify recombinant TADG-15 cDNA clones, and todistinguish them from other cDNA clones.

Further included in this invention are TADG-15 proteins which areencoded, at least in part, by portions of SEQ ID No. 2, e.g., productsof alternative mRNA splicing or alternative protein processing events,or in which a section of TADG-15 sequence has been deleted. Thefragment, or the intact TADG-15 polypeptide, may be covalently linked toanother polypeptide, e.g., one which acts as a label, a ligand or ameans to increase antigenicity.

The invention also includes a polyclonal or monoclonal antibody whichspecifically binds to TADG-15. The invention encompasses not only anintact monoclonal antibody, but also an immunologically-active antibodyfragment, e.g., a Fab or (Fab)₂ fragment; an engineered single chain Fvmolecule; or a chimeric molecule, e.g., an antibody which contains thebinding specificity of one antibody, e.g., of murine origin, and theremaining portions of another antibody, e.g., of human origin.

In one embodiment, the antibody, or a fragment thereof, may be linked toa toxin or to a detectable label, e.g., a radioactive label,non-radioactive isotopic label, fluorescent label, chemiluminescentlabel, paramagnetic label, enzyme label, or colorimetric label. Examplesof suitable toxins include diphtheria toxin, Pseudomonas exotoxin A,ricin, and cholera toxin. Examples of suitable enzyme labels includemalate hydrogenase, staphylococcal nuclease, delta-5-steroid isomerase,alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triosephosphate isomerase, peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase, acetylcholinesterase,etc. Examples of suitable radioisotopic labels include ³H, ¹²⁵I, ¹³¹I,³²P, ³⁵S, ¹⁴C. etc.

Paramagnetic isotopes for purposes of in vivo diagnosis can also be usedaccording to the methods of this invention. There are numerous examplesof elements that are useful in magnetic resonance imaging. Fordiscussions on in vivo nuclear magnetic resonance imaging, see, forexample, Schaefer et al., (1989) JACC 14, 472-480; Shreve et al., (1986)Magn. Reson. Med. 3, 336-340; Wolf. G. L., (1984) Physiol. Chem. Phys.Med. NMR 16, 93-95; Wesbey et al., (1984) Physiol. Chem. Phys. Med. NMR16, 145-155; Runge et al., (1984) Invest. Radiol. 19, 408-415. Examplesof suitable fluorescent labels include a fluorescein label, anisothiocyalate label, a rhodamine label, a phycoerythrin label, aphycocyanin label, an allophycocyanin label, an ophthaldehyde label, afluorescamine label, etc. Examples of chemiluminescent labels include aluminal label, an isoluminal label, an aromatic acridinium ester label,an imidazole label, an acridinium salt label, an oxalate ester label, aluciferin label, a luciferase label, an aequorin label, etc.

Those of ordinary skill in the art will know of other suitable labelswhich may be employed in accordance with the present invention. Thebinding of these labels to antibodies or fragments thereof can beaccomplished using standard techniques commonly known and used by thoseof ordinary skill in the art. Typical techniques are described byKennedy et al. (1976) Clin. Chim. Acta 70. 1-31 and Schurs et al. (1977)Clin. Chim. Acta 81, 1-40. Coupling techniques mentioned in the latterare the glutaraldehyde method, the periodate method, the dimaleimidemethod, the m-maleimidobenzyl-N-hydroxy-succinimide ester method. All ofthese methods are incorporated by reference herein.

Also within the invention is a method of detecting TADG-15 protein in abiological sample, which includes the steps of contacting the samplewith the labeled antibody, e.g., radioactively tagged antibody specificfor TADG-15, and determining whether the antibody binds to a componentof the sample. Antibodies to the TADG-15 protein can be used in animmunoassay to detect increased levels of TADG-15 protein expression intissues suspected of neoplastic transformation. These same uses can beachieved with Northern blot assays and analyses.

As described herein, the invention provides a number of diagnosticadvantages and uses. For example, the TADG-15 protein is useful indiagnosing cancer in different tissues since this protein is highlyoverexpressed in tumor cells. Antibodies (or antigen-binding fragmentsthereof) which bind to an epitope specific for TADG-15, are useful in amethod of detecting TADG-15 protein in a biological sample for diagnosisof cancerous or neoplastic transformation. This method includes thesteps of obtaining a biological sample (e.g., cells, blood, plasma,tissue, etc.) from a patient suspected of having cancer, contacting thesample with a labeled antibody (e.g., radioactively tagged antibody)specific for TADG-15, and detecting the TADG-15 protein using standardimmunoassay techniques such as an ELISA. Antibody binding to thebiological sample indicates that the sample contains a component whichspecifically binds to an epitope within TADG-15.

Likewise, a standard Northern blot assay can be used to ascertain therelative amounts of TADG-15 mRNA in a cell or tissue obtained from apatient suspected of having cancer, in accordance with conventionalNorthern hybridization techniques known to those of ordinary skill inthe art. This Northern assay uses a hybridization probe, e.g.,radiolabelled TADG-15 cDNA, either containing the full-length, singlestranded DNA having a sequence complementary to SEQ ID No. 1 (FIGS.2A-2D), or a fragment of that DNA sequence at least 20 (preferably atleast 30, more preferably at least 50, and most preferably at least 100consecutive nucleotides in length). The DNA hybridization probe can belabeled by any of the many different methods know to those skilled inthis art.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion.

EXAMPLE 1 Tissue Collection and Storage

Upon patient hysterectomy, bilateral salpingo-oophorectomy, or surgicalremoval of neoplastic tissue, the specimen is retrieved and placed onice. The specimen was then taken to the resident pathologist forisolation and identification of specific tissue samples. Finally, thesample was frozen in liquid nitrogen, logged into the laboratory recordand stored at −80° C.

Additional specimens were frequently obtained from the Cooperative HumanTissue Network (CHTN). These samples were prepared by the CHTN andshipped on dry ice. Upon arrival, these specimens (e.g., blood (serum),urine, saliva, tears and insterstitial fluid) were logged into thelaboratory record and stored at −80° C. Participation of the followingdivisions of the Cooperative Human Tissue Network (CHTN) in providingtumor tissues is acknowledged: Western Division, Case Western ReserveUniversity, (Cleveland, Ohio); Midwestern Division, Ohio stateUniversity, (Columbus, Ohio); Eastern Division, NDRI, (Philadelphia.Pa.); Pediatric Division, Children's Hospital, (Columbus, Ohio);Southern Division, University of Alabama at Birmingham, (Birmingham,Ala.).

EXAMPLE 2 mRNA Isolation and cDNA Synthesis

Forty-one ovarian tumors (10 low malignant potential tumors and 31carcinomas) and 10 normal ovaries were obtained from surgical specimensand frozen in liquid nitrogen. The human ovarian carcinoma cell linesSW626 and CAOV3, and the human breast carcinoma cell lines MDA-MB-231and MDA-MB-435S, were purchased from the American Type CultureCollection (Rockville, Md.). Cells were cultured to sub-confluency inDulbecco's modified Eagle's medium supplemented with 10% (v/v) fetalbovine serum and antibiotics.

Messenger RNA (mRNA) isolation was performed according to themanufacturer's instructions using the Mini RiboSep™ Ultra mRNA IsolationKit purchased from Becton Dickinson. In this procedure, polyA⁺ mRNA wasisolated directly from the tissue lysate using the affinitychromatography media oligo(dT) cellulose. The amount of mRNA recoveredwas quantitated by UV spectrophotometry.

First-strand complementary DNA (cDNA) was synthesized using 5.0 μg ofmRNA and either random hexamer or oligo(dT) primers according to themanufacturer's protocol utilizing a first strand synthesis kit obtainedfrom CLONTECH (Palo Alto, Calif.). The purity of the cDNA was evaluatedby PCR using primers specific for the p53 gene. These primers span anintron such that pure cDNA can be distinguished from cDNA that iscontaminated with genomic DNA.

EXAMPLE 3 PCR With Redundant Primers, Cloning of TADG-15 cDNA. T-vectorLigation and Transformations and DNA Sequencing

Redundant primers, forward 5′-TGGGTIGTIACIGCIGCICA(C/T)TG-3′ (SEQ ID No.11) and reverse 5′-A(A/G)IGGICCICCI(C/G)(T/A)(A/G)TCICC-3′ (SEQ ID No.12), corresponding to the amino acids surrounding the catalytic triadfor serine proteases, were used to compare the PCR products from normaland carcinoma cDNAs.

The purified PCR products were ligated into the Promega T-vector plasmidand the ligation products used to transform JM109 competent cellsaccording to the manufacturer's instructions (Promega). Positivecolonies were cultured for amplification, the plasmid DNA isolated usingthe Wizard™ Minipreps DNA purification system (Promega), and theplasmids were digested with ApaI and SacI restriction enzymes todetermine the size of the insert. Plasmids with inserts of the size(s)visualized by the previously described PCR product gel electrophoresiswere sequenced.

Individual colonies were cultured and plasmid DNA was isolated using theWizard Miniprep DNA purification system (Promega). Applied BiosystemsModel 373A DNA sequencing system was used for direct cDNA sequencedetermination. Utilizing a plasmid-specific primer near the cloningsite, sequencing reactions were carried out using PRISM™ Ready ReactionDye Deoxy™ terminators (Applied Biosystems) according to themanufacturer's instructions. Residual dye terminators were removed fromthe completed sequencing reaction using a Centri-sep™ spin column(Princeton Separation). Based upon the determined sequence. primers thatspecifically amplify the gene of interest were designed and synthesized.

The original TADG-15 subclone (436 bp) was randomly labeled and used asa probe to screen an ovarian tumor cDNA library by standardhybridization techniques.¹³ The library was constructed in 8ZAP usingmRNA isolated from the tumor cells of a stage III/grade III ovarianadenocarcinoma patient. Three overlapping clones were obtained whichspanned 3147 nucleotides.

EXAMPLE 4 Northern Blot Analysis

10 μg mRNAs were size separated by electrophoresis through a 1%formaldehyde-agarose gel in 0.02 M MOPS, 0.05 M sodium acetate (pH 7.0),and 0.001 M EDTA. The mRNAs were then blotted to Hybond-N⁺ nylonmembrane (Amersham) by capillary action in 20×SSPE. The RNAs are fixedto the membrane by baking for 2 hours at 80° C. ³²P-labeled cDNA probeswere made by Prime-a-Gene Labeling System (Promega). The PCR productsamplified by the same primers described above were used for probes. Theblots were prehybridized for 30 min and hybridized for 60 min at 68° C.with ³²P-labeled cDNA probe in ExpressHyb Hybridization Solution(CLONTECH). Control hybridization to determine relative gel loading wasperformed with a β-tubulin probe.

Normal human tissues; spleen, thymus, prostate, testis, ovary, smallintestine, colon and peripheral blood leukocyte, and normal human fetaltissues; brain, lung, liver and kidney (Human Multiple Tissue NorthernBlot; CLONTECH) were also examined by the same hybridization procedure.Additional multiple tissue northern (MTN) blots from CLONTECH includethe Human MTN blot, the Human MTN II blot, the Human Fetal MTN II blot,and the Human Brain MTN III blot.

EXAMPLE 5 Western Blot Analysis

Polyclonal rabbit antibody was generated by immunization with apoly-lysine linked multiple Ag peptide derived from the TADG-15 proteinsequence ‘LFRDWIKENTGV’ (SEQ ID No. 13). Approximately 20 μg of celllysates were separated on a 15% SDS-PAGE gel and electroblotted to PVDFat 100 V for 40 min at 4° C. The proteins were fixed to the membrane byincubation in 50% MeOH for 10 min. The membrane was blocked overnight inTBS (pH 7.8) containing 0.2% non-fat milk. Primary antibody was added tothe membrane at a dilution of 1:100 in 0.2% milk/TBS and incubated for 2h at room temperature. The blot was washed and incubated with a 1:3000dilution of alkaline-phosphatase conjugated goat anti-rabbit IgG(BioRad) for 1 h at room temperature. The blot was washed and incubatedwith a chemiluminescent substrate before a 10 sec exposure to X-ray filmfor visualization.

EXAMPLE 6 Quantitative PCR

The mRNA overexpression of TADG-15 was determined using a quantitativePCR. Quantitative PCR was performed. ^(11.12) Oligonucleotide primerswere used for TADG-15:

forward 5′ATGACAGAGGATTCAGGTAC-3′ (SEQ ID No. 14) and

reverse 5′GAAGGTGAAGTCATTGAAGA-3′ (SEQ ID No. 15); and and forβ-tubulin:

forward 5′CGCATCAACGTGTACTACAA-3′ (SEQ ID No.16) and

reverse 5′TACGAGCTGGTGGACTGAGA-3′ (SEQ ID No. 17), β-tubulin wasutilized as an internal control.

The PCR reaction mixture consists of cDNA derived from 50 ng of mRNA, 5pmol of sense and antisense primers for both the TADG-15 gene and theβ-tubulin gene, 200 μmol of dNTPs, 5 μCi of α-³²PdCTP and 0.25 units ofTaq DNA polymerase with reaction buffer (Promega) in a final volume of25 μl. The target sequences were amplified in parallel with theβ-tubulin gene. Thirty cycles of PCR were carried out in a ThermalCycler (Perkin Elmer Gene Amp 2400; Perkin-Elmer Cetus). Each cycle ofPCR included 30 sec of denaturation at 94° C., 30 sec of annealing at60° C. and 30 sec of extension at 72° C. The annealing temperaturevaries according to the primers that are used in the PCR reaction. Forthe reactions involving degenerate primers, an annealing temperature of48° C. was used. The appropriate annealing temperature for the TADG-15-and β-tubulin-specific primers is 62° C.

A portion of the PCR products were separated on 2% agarose gels and theradioactivity of each PCR product was determined by using aPhosphoImager (Molecular Dynamics). In the present study, the expressionratio (TADG-15/β-tubulin) was used to evaluate gene expression anddefined the value at mean ±2SD of normal ovary as the cut-off value todetermine overexpression. The student's t test was used for comparisonof the mean values of normal ovary and tumors.

EXAMPLE 7 Immunohistochemistry

Immunohistochemical staining was performed using a Vectastain Elite ABCKit (Vector). Formalin-fixed and paraffin-embedded specimens wereroutinely deparaffinized and processed using microwave heat treatment in0.01 M sodium citrate buffer (pH 6.0). The specimens were incubated withnormal goat serum in a moist chamber for 30 min. After incubation withbiotinylated anti-rabbit IgG for 30 min, the sections were thenincubated with ABC reagent (Vector) for 30 min. The final products werevisualized using the AEC substrate system (DAKO) and sections werecounterstained with hematoxylin before mounting. Negative controls wereperformed using normal serum instead of the primary antibody.

EXAMPLE 8 Antisense TADG-15

TADG-15 is cloned and expressed in the opposite orientation such that anantisense RNA molecule (SEQ ID No. 18) is produced. For example, theantisense RNA is used to hybridize to the complementary RNA in the celland thereby inhibit translation of TADG-15 RNA into protein.

EXAMPLE 9 Peptide Ranking

For vaccine or immune stimulation, individual 9-mers to 11-mers wereexamined to rank the bindings of individual peptides to the top 8halplotypes in the general population (Parker et al. (1994)). Thecomputer program used for this analyses can be found at<http://www-bimas.dert.nih.gov/molbio/hla_bind/>. Table 1 shows thepeptide ranking based upon the predicted half-life of each peptide'sbinding,y to a particular HLA allele. A larger half-life indicates astronger association with that peptide and the particular HLA molecule.The TADG-15 peptides that strongly bind to an HLA allele are putativeimmunogens, and are used to innoculate an individual against TADG-15.

TABLE 1 TADG-15 peptide ranking HLA Type Predicted SEQ & Ranking StartPeptide Dissociation_(½) ID No. HLA A0201 1 68 VLLGIGFLV 2537.396 19 2126 LLYSGVPFL 1470.075 20 3 644 SLISPNWLV 521.640 21 4 379 KVSFKFFYL396.525 22 5 386 YLLEPGVPA 346.677 23 6 257 SLTFRSFDL 123.902 24 7 762ILQKGEIRV 118.238 25 8 841 RLPLFRDWI 106.842 26 9 64 GLLLVLLGI 88,783 2710  57 VLAAVLIGL 83.527 28 HLA A0205 1 67 LVLLGIGFL 142.800 29 2 379KVSFKFFYL 100.800 30 3 126 LLYSGVPFL 71.400 31 4 88 KVFNGYMRI 36.000 325 670 TQWTAFLGL 33.600 33 6 119 KVKDALKLL 25.200 34 7 60 AVLIGLLLV24.000 35 8 62 LIGLLLVLL 23.800 36 9 57 VLAAVLIGL 23.800 37 10  61VLIGLLLVL 23.800 38 HLA A1 1 146 FSEGSVIAY 337.500 39 2 658 YIDDRGFRY125.000 40 3 449 SSDPCPGQF 75.000 41 4 401 YVEINGEKY 45.000 42 5 387LLEPGVPAG 18.000 43 6 553 GSDEASCPK 15.000 44 7 97 TNENFVDAY 11.250 45 8110 STEFVSLAS 11.250 46 9 811 SVEADGRIF 9.000 47 10  666 YSDPTQWTA 7.50048 HLA A24 1 709 DYDIALLEL 220.000 49 2 408 KYCGERSQF 200.000 50 3 754QYGGTGALI 50.000 51 4 153 AYYWSEFSI 50.000 52 5 722 EYSSMVRPI 50.000 536 326 GFEATFFQL 36.000 54 7 304 TFHSSQNVL 24.000 55 8 707 TFDYDIALL20.000 56 9 21 KYNSRHEKV 16.500 57 10  665 RYSDPTQWT 14.400 58 HLA B7 1686 APGVQERRL 240.000 59 2 12 GPKDFGAGL 80.000 60 3 668 DPTQWTAFL 80.00061 4 461 TGRCIRKEL 60.000 62 5 59 AAVLIGLLL 36.000 63 6 379 KVSFKFFYL20.000 64 7 119 KVKDALKLL 20.000 65 8 780 LPQQITPRM 20.000 66 9 67LVLLGIGFL 20.000 67 10  283 SPMEPHALV 18.000 68 HLA B8 1 12 GPKDFGAGL24.000 69 2 257 SLTFRSFDL 8.000 70 3 180 MLPPRARSL 8.000 71 4 217GLHARGVEL 8.000 72 5 173 MAEERVVML 4.800 73 6 267 SCDERGSDL 4.800 74 7567 CTKHTYRCL 4.000 75 8 724 SSMVRPICL 4.000 76 9 409 YCGERSQFV 3.600 7710  495 TCKNKFCKP 3.200 78 HLA B2702 1 427 VRFHSDQSY 1000.000 79 2 695KRIISHPFF 600.000 80 3 664 FRYSDPFQW 500.000 81 4 220 ARCVELMRF 200.00082 5 492 HQFTCKNTKF 100.000 83 6 53 GRWVVLAAV 100.000 84 7 248 LRGDADSVL60.000 85 8 572 YRCLNGLCL 60.000 86 9 692 RRLKRIISH 60.000 87 10  24SRHEKVNGL 60.000 88 HLA B4403 1 147 SEGSVLAYY 360.000 89 2 715 LELEKPAEY360.000 90 3 105 YENSNSThF 60.000 91 4 14 KDFGAGLKY 50.625 92 5 129SGVPFLCPY 36.000 93 6 436 TDTGFLAEY 33.750 94 7 766 GEIRVINQT 30.000 958 402 VEINGEKYC 30.000 96 9 482 DELNCSCDA 24.000 97 10  82 RDVRVQKVF22.500 98

EXAMPLE 10 TADG-15cDNA

A screening strategy to identify proteases which are overexpressed inhuman cancer has been developed in which RT-PCR products amplifiedspecific in tumors, as compared to normal tissue, are examined.⁹ Duringthis effort, candidate genes were identified using redundant senseprimers to the conserved amino acid histidine domain at the NH₃ end ofthe catalytic domain and antisense primers to the downstream conservedamino acid serine domain. Subcloning and sequencing the appropriate 480base pair band(s) amplified in such a PCR reaction provides the basisfor identifying the gene(s) encoding proteases(s). Among these amplifiedcatalytic domains, a new serine protease gene named TADG-15 (tumorantigen-derived gene 15) was identified. The catalytic domain of thenewly identified TADG-15 protein is similar to other serine proteasesand specifically contains conserved amino acids appropriate for thecatalytic domain of the trypsin-like serine protease family.

A computerized search of GenEMBL databases using the FASTA program(Wisconsin Package Version 9.1, GCG, Madison, Wis.) for amino acidsequences homologous to the TADG-15 protease domain revealed thathomologies with other known human proteases never exceeds 55%. FIGS. 1Aand 1B show the alignment of the protease domain of TADG-15 comparedwith other human serine proteases. Using the BESTFIT program, availablethrough GCG, the similarities between TADG-15 and trypsin, chymotrypsin,and tissue-type plasminogen activator are 51%, 46% and 52%,respectively.

From the sequence derived from the TADG-15 catalytic domain, specificprimers were synthesized to amplify a TADG-15-specific probe for libraryscreening. After screening an ovarian carcinoma library, one 1785 bpclone was obtained which included the 3″ end of the TADG-15 transcript.Upon further screening using the 5″ end of the newly detected clone, twoadditional clones were identified which provided another 1362 bp of thecDNA, including the 5′ end of the TADG-15 transcript. The total lengthof the sequenced cDNA was approximately 3.15 kb. The total nucleotidesequence obtained includes a Kozak's consensus sequence preceding asingle open reading frame encoding a predicted protein of 855 aminoacids (FIGS. 2A-2D).

The deduced open reading frame encoded by the TADG-15 nucleotidesequence (FIGS. 2A-2D, 3 and 4) contains several distinct domains asfollows: an amino terminal cytoplasmic tail (amino acids (aa) #1-54), apotential transmembrane domain (aa #55-77), an extracellular membranedomain (aa #78-213), two complement subcomponents Clr/Cls, Uegf, andbone morphogenetic protein 1 (CUB) repeats (aa #214-447), four ligandbinding repeats of the low density lipoprotein (LDL) receptor-likedomain (aa #453-602) and a serine protease domain (aa #615-855). TheTADG-15 protein also contains two potential N-linked glycosylation sites(aa #109 and 302) and a potential proteolytic cleavage site upstreamfrom the protease domain (aa #614) which could release and/or activatethe protease at the carboxy end of this protein. In addition, TADG-15contains an RGD motif (aa #249-251) which is commonly found in proteinsinvolved in cell-cell adhesion.

EXAMPLE 11 TADG-15 Expression

To examine the size of the transcript for TADG-15 and its pattern ofexpression in various tissues, Northern blot hybridization was performedfor representative histological types of carcinoma and in a series ofcell lines, fetal tissues and normal adult tissues (FIG. 5). Thetranscript size for the TADG-15 message was determined to beapproximately 3.2 kb and a single intense transcript appeared to bepresent in all of the carcinomas examined, whereas no visible band wasdetected in normal ovary (FIG. 5). This transcript size is also in goodagreement with the sequence data predicting a transcript size of 3.15kb. The ovarian tumor cell lines. SW626 and CAOV3, also showed anabundance of transcript, however little or no transcript was detectablein the breast carcinoma cell lines MDA-MB-231 and MDA-MB-4355. Amongnormal human fetal tissues, fetal kidney showed an abundance of theTADG-15 transcript and low expression was also detected in fetal lung.In normal adult tissues. TADG-15 was detected in colon with low levelsof expression in small intestine and prostate (FIG. 5).

To evaluate mRNA transcript expression of TADG-15 in ovarian tumors andnormal ovary, semi-quantitative PCR (FIG. 6) was performed. In apreliminary study, the linearity of this assay^(11.12) was confirmed andits efficacy correlated with both Northern blots andimmunohistochemistry. The data was quanitified using a phosphoimager andcompared as a ratio of expression (TADG-15/β-tubulin). Results hereinindicate that TADG-15 transcript expression is elevated above thecut-off value (mean for normal ovary ±2 SD) in all of the tumor casesexamined and is either not detected or detected at extremely low levelsin normal ovaries (FIG. 6A and B). Analysis of ovarian carcinomasubtypes, including early stage and late stage disease, confirmsoverexpression of TADG-15 in all carcinomas examined (Table 2). All ofthe carcinomas studied, which included 5 stage I and 3 stage IIcarcinomas, showed overexpression of the TADG-15 gene.

These data can also be examined with regard to tumor stage andhistological sub-type, and results indicated that every carcinoma ofevery stage and histological sub-type overexpressed the TADG-15 gene.The expression ratio (mean value ±SD) for normal ovary group wasdetermined as 0.182±0.024, for LMP tumor group as 0.847±0.419 and forcarcinoma group as 0.771±0.380 (Table 2). A comparison between thenormal ovary group and tumor groups showed that overexpression of theTADG-15 gene is statistically significant in both the LMP tumor groupand the carcinoma group (LMP tumor: p<0.001, carcinoma: p<0.0001).

As shown in FIG. 6, TADG-15 transcripts were noted in all ovariancarcinomas, but were not present at detectable levels in any of thefollowing tissues: a) normal ovary, b) fetal liver and brain, c) adultspleen, thymus, testes, ovary and peripheral blood lymphocytes, d)skeletal muscle, liver, brain or heart. This evaluation was extended toa standard panel of about 40 tumors. Using TADG-15-specific primers, theexpression was also examined in tumor cell lines derived from bothovarian and breast carcinoma tissues as shown in FIG. 7 and in othertumor tissues as shown in FIG. 8. Expression of TADG-15 was alsoobserved in carcinomas of the breast, colon, prostate and lung.

Polyclonal antibodies developed to a synthetic peptide (a 12-mer) at thecarboxy terminus of the protease domain were used to examine TADG-15expression in cell lines by Western blot and by immunolocalization innormal ovary and ovarian tumors. Western blots of cell extracts fromSW626 and CAOV3 cells were probed with both antibody and preimmune sera(FIG. 9). Several bands were detected with the antibody, including bandsof approximately 100,000 daltons, approximately 60,000 daltons and32,000 daltons. The anticipated molecular size of the complete TADG-15molecule is estimated to be approximately 100,000 daltons, and theprotease domain which may be released by proteolytic cleavage at aa #614is estimated to be approximately 32,000 daltons. Some intermediateproteolytic product may be represented by the 60,000 dalton band.

Antibody staining of tumor cells confirms the presence of the TADG-15protease in the cytoplasm of a serous LMP tumor, mucinous LMP tumor andserous carcinoma (FIG. 10B, C & D, respectively). This diffuse stainingpattern may be due to detection of TADG15 within the cell as it is beingpackaged and transported to the cell surface. In endometrioid carcinoma,the antigen is clearly detectable on the surface of tumor cells (FIG.10E). No staining was detected in normal ovarian epithelium or stromalcells (FIG. 10A). Immunohistochemical staining of a series of 27 tumorsindicates the presence of the TADG-15 protein in all the carcinomasubtypes examined, including the low malignant potential group. Strongstaining was noted in 7 of 9 low malignant potential tumors and 13 of 18carcinomas (Table 3).

TABLE 2 Number of cases with overexpression of TADG-15 in normal ovariesand ovarian tumors overexpression N of TADG-15 expression ratio^(a)Normal 10 0 (0%)  0.182 ± 0.024 LMP 10 10 (100%)  0.847 ± 0.419 serous 66 (100%) 0.862 ± 0.419 mucinous 4 4 (100%) 0.825 ± 0.483 Carcinoma 31 31(100%)  0.771 ± 0.380 serous 18 18 (100%)  0.779 ± 0.332 mucinous 7 7(100%) 0.907 ± 0.584 endometrioid 3 3 (100%) 0.502 ± 0.083 clear cell 33 (100%) 0.672 ± 0.077 ^(a)The ratio of expression level of TADG-15 toβ-tubulin (mean ± SD)

TABLE 3 Immunohistochemical staining using TADG-15 Lab No. HistologyTADG-15 Surface epithelium of the ovary − H-3194 serous (LMP) ++ H-162scrous (LMP) ++ H-1182 serous (LMP) ++ H-4818 serous (LMP) ++ H-4881serous (LMP) ++ H-675 mucinous (LMP) + H-2446 mucinous (LMP) + H-0707mucinous (LMP) ++ H-2042 mucinous (LMP) ++ H-2555 serous carcinoma ++H-1858 serous carcinoma ++ H-5266 serous carcinoma ++ H-5316 serouscarcinoma + H-2597 serous carcinoma + H-4931 mucinous carcinoma ++H-1867 mucinous carcinoma ++ H-5998 mucinous carcinoma ++ H-2679endometrioid adenocarcinoma + H-5718 endometrioid adenocarcinoma ++H-3993 endometrioid adenocarcinoma + H-2991 endometrioid adenocarcinoma++ H-2489 endometrioid adenocarcinoma ++ H-5994 clear cell carcinoma ++H-6718 clear cell carcinoma ++ H-1661 clear cell carcinoma ++ H-6201clear cell carcinoma ++ H-5640 clear cell carcinoma + − Negative; + WeakPositive; ++ Strong Positive (more than 50% of cell staining)

EXAMPLE 12 TADG-15 Homology

Recently, a mouse protein named epithin (GenBank Accession No. AF04282)has been described.¹⁴ Epithin is a 902 amino acid protein which containsa similar structure to TADG-15 in that it has a cytoplasmic domain,transmembrane domain, two CUB domains, four LDLR-like domains and acarboxy terminal serine protease domain. TADG-15 and epithin are 84%similar over 843 amino acids, suggesting that the proteins may beorthologous (FIGS. 11A and 11B). The precise role of epithin remains tobe elucidated.

A search of GeneBank for similar previously identified sequences yieldedone such sequence with relatively high homology to a portion of TADG-15from nucleotide #182 to 3139 and SNC-19 GeneBank Accession No. #U20428)is approximately 97% (FIGS. 12A-12E). There are however significantdifferences between SNC-19 and TADG-15. For example, TADG-15 has an openreading frame of 855 amino acids whereas the longest open reading frameof SNC-19 is 173 amino acids. Additionally, SNC-19 does not include aproper start site for the initiation of translation, nor does it includethe amino terminal portion of the protein encoded by TADG-15. Moreover,SNC-19 does not include an open reading frame for a functional serineprotease because the His, Asp and Ser residues of the catalytic triadthat are necessary for function are encoded in different reading frames.

Implications

The overall structure of the TADG-15 protein is relatively similar tothe members of the tolloid/BMP-1 family and the complementsubcomponents, Clr/Cls. These proteins contain both CUB and proteasedomains, and complex formation through the ligand binding domain isessential for their function. Activation of the serine protense domainsof Clr and Cls requires proteolytic cleavage of Arg-Gly and Arg-Ilebonds, respectively.¹⁵ Similarly, it might be expected that the TADG-15protein is synthesized as a zymogen, which is activated by cleavagebetween Arg⁶¹⁴ and Val⁶¹⁵ and analogous to the activation mechanism ofother serine protease zymogens. Western blot analysis of cultured celllysates confirmed both a 100 kDa and 32 kDa peptide, which correspond tothe putative zymogen (whole molecule) and a cleaved protease product ofTADG-15 (FIG. 9). These data support a model for proteolytic releaseand/or activation of TADG-15 as occurs for similar type II serineproteases.

CUB domains were first found in complement subcomponents Clr/Cls¹⁶⁻¹⁸and are known to be a widespread module in developmentally regulatedproteins, such as the bone morphogenetic protein-1 (BMP-1) and thetolloid gene product.¹⁸⁻²⁰ The role of these repeats remains largelyunknown. However, some models suggest that the CUB domain may beinvolved in protein-protein interactions. The CUB domain of Clr and Clsparticipates in the assembly of the Cls-Clr-Clr-Cls tetrameric complexin the activation of the classical pathway of complement by providingprotein-protein interaction domains.¹⁵ The Drosophila decapentaplegic(DPP) protein is essential for dorsal-ventral specification of theembryo, and the Drosophila tolloid (TLD) forms a complex with DPP toregulate its activity.¹⁹⁻²⁰ Missense mutations in the CUB domain of thetolloid protein results in a phenotype that does not allow a proteininteraction with the DPP complex.¹⁹

The TADG-15 protein contains two tandem repeats of CUB-like domainsbetween amino acid residues 214 and 447. Each of these is approximately110 amino acids long and each has four conserved cysteine residuescharacteristic of other CUBs (amino acids 214, 244, 268, 294, 340, 366,397, 410). By analogy, the CUB repeats of the TADG-15 protein may forman interactive domain capable of promoting multimeric complex formationand regulating the activity of the target protein or TADG-15 itself.

The TADG-15 protein also contains the LDL receptor ligand binding repeat(class A motif) -like domain, which consists of four contiguouscysteine-rich repeats (amino acid residues 453 to 602). Eachcysteine-rich repeat is approximately 40 amino acids long and contains aconserved, negatively-charged sequence (Ser-Asp-Glu) with six cysteineresidues. In the LDL receptor protein, this repeat is thought tofunction as a protein-binding domain which interacts with the lysine andarginine residues present in lipoproteins.^(21.22) In addition, thefirst repeat of the LDL receptor appears to bind Ca²⁺ and not thelipoproteins.²³ By analogy, it is possible that the LDL receptor-likerepeat in TADG-15 may act in a similar fashion, interacting withpositively charged regions of other proteins and/or as a Ca²⁺ binding,site. As a result of ligand binding and the formation of receptor-ligandcomplex, LDL receptor is internalized via clathrin-coated pits.²⁴ Thesetypes of plasma membrane receptors contain a characteristic amino acidsequence in their cytoplasmic domain for binding to clathrin-coatedpits.²⁴ TADG-15 does not contain this motif in its cytosolic region, andfurthermore, no similarities with other known protein sequences werefound in the cytoplasmic domain of the TADG-15. This finding suggeststhat TADG-15 functions in a different manner from the endocyticreceptors (such as the LDL receptor), although TADG-15 possesses similarligand-binding repeats in the extracellular matrix.

Although the precise role of TADG-15 is unknown, this gene is clearlyoverexpressed in ovarian tumors. A variety of proteases, such as type IVcollagenase and plasminogen activator, appear to be involved in theprocess of tumor invasion and are constituents of a protease cascade inmalignant progression. TADG-15 may constitute such an activity anddirectly digest extracellular matrix components surrounding a tumor, oractivate other proteases by cleavage of inactive precursors, indirectlyenhancing tumor growth and invasion. It is also possible that TADG-15may function like a member of the tolloid/BMP-1 family by formingcomplexes with other growth factors or signal transduction proteins tomodulate their activities.

These data raise the possibility that the TADG-15 gene and itstranslated protein will be a useful marker for the early detection ofovarian carcinoma through release of the protease domain into theextracellular matrix and ultimately the circulation. These data alsosuggest the possibility of using TADG-15 as a target for therapeuticintervention through delivery systems directed at the CUB/LDLR ligandbinding domains.

The following references were cited herein:

1. Liotta, L. A., et al. Cell, 64: 327-336, 1991.

2. Duffy, M. J. Clin. Exp. Metastasis. 10: 145-155, 1992.

3. Tryggvason. K., et al. Biochem. Biophys. Acta., 907: 191-217, 1987.

4. Levy, A. T., et al. Cancer Res., 51: 439-444, 1991.

5. Monsky, W. L. et al. Cancer Res., 53: 3159-3164, 1993.

6. Duffy, M. J. et al. Cancer, 62: 531-533, 1988.

7. Häckel, C. et al. Cancer, 79: 53-58, 1997.

8. Watt. K. et al. Proc. Natl. Acad. Sci. U.S.A., 83: 3166-3170. 1986.

15 9. Tanimoto, H. et al. Cancer Res., 57: 2884-2887, 1997.

11. Shigemasa, K. et al. J. Soc. Gynecol. Invest., 4: 95-102, 1997.

12. Tanimoto, H. et al. Gynecol. Oncol., 66: 308-312, 1997.

13. Maniatis, T. Fritsch, E. F. & Sambrook, J. Molecular Cloning, p.309-361 Cold Spring Harbor Laboratory, New York, 1982.

14. Kim, M. G., et al. Immunogenetics, 49(5): 420-428, 1999.

15. Arlaud et al. Method in Enzymology, 223: 61-82, 1993.

16. Journet, A. & Tosi, M. Biochem. J., 240: 783-787, 1986.

17. Mackinnon, C. M., et al. Eur. J. Biochem., 169: 547-553, 1987.

18. Bork, P. & Beckmann, G. J. Mol. Biol., 231: 539-545, 1993.

19. Childs, S. R. & O'Connor, M. B. Dev. Biol., 162: 209-220, 1994.

20. Blader, P L. et al. Science, 278: 1937-1940, 1997.

21. Yamamoto, T. et al. Cell, 39: 27-38, 1984.

22. Daly, N. L. et al. Proc. Natl. Acad. Sci., 92: 6334-6338, 1995.

23. van Driel, I. R. et al. J. Biol. Chem., 262: 17443-17449, 1987.

24. Lodish, H. et al. Sorting of membrane proteins internalized from thecell surface. In: Molecular Cell Biology, 3rd ed., p.722-733 ScientificAmerican Books, Inc., New York, 1995.

25. Parker, K C et al. J. Immunol. 152:163, 1994.

Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

98 1 3147 DNA Homo sapiens TADG-15 1 tcaagagcgg cctcggggta ccatggggagcgatcgggcc cgcaagggcg gagggggccc 60 gaaggacttc ggcgcgggac tcaagtacaactcccggcac gagaaagtga atggcttgga 120 ggaaggcgtg gagttcctgc cagtcaacaacgtcaagaag gtggaaaagc atggcccggg 180 gcgctgggtg gtgctggcag ccgtgctgatcggcctcctc ttggtcttgc tggggatcgg 240 cttcctggtg tggcatttgc agtaccgggacgtgcgtgtc cagaaggtct tcaatggcta 300 catgaggatc acaaatgaga attttgtggatgcctacgag aactccaact ccactgagtt 360 tgtaagcctg gccagcaagg tgaaggacgcgctgaagctg ctgtacagcg gagtcccatt 420 cctgggcccc taccacaagg agtcggctgtgacggccttc agcgagggca gcgtcatcgc 480 ctactactgg tctgagttca gcatcccgcagcacctggtg gaggaggccg agcgcgtcat 540 ggccgaggag cgcgtagtca tgctgcccccgcgggcgcgc tccctgaagt cctttgtggt 600 cacctcagtg gtggctttcc ccacggactccaaaacagta cagaggaccc aggacaacag 660 ctgcagcttt ggcctgcacg cccgcggtgtggagctgatg cgcttcacca cgcccggctt 720 ccctgacagc ccctaccccg ctcatgcccgctgccagtgg gccctgcggg gggacgccga 780 ctcagtgctg agcctcacct tccgcagctttgaccttgcg tcctgcgacg agcgcggcag 840 cgacctggtg acggtgtaca acaccctgagccccatggag ccccacgccc tggtgcagtt 900 gtgtggcacc taccctccct cctacaacctgaccttccac tcctcccaga acgtcctgct 960 catcacactg ataaccaaca ctgagcggcggcatcccggc tttgaggcca ccttcttcca 1020 gctgcctagg atgagcagct gtggaggccgcttacgtaaa gcccagggga cattcaacag 1080 cccctactac ccaggccact acccacccaacattgactgc acatggaaca ttgaggtgcc 1140 caacaaccag catgtgaagg tgagcttcaaattcttctac ctgctggagc ccggcgtgcc 1200 tgcgggcacc tgccccaagg actacgtggagatcaatggg gagaaatact gcggagagag 1260 gtcccagttc gtcgtcacca gcaacagcaacaagatcaca gttcgcttcc actcagatca 1320 gtcctacacc gacaccggct tcttagctgaatacctctcc tacgactcca gtgacccatg 1380 cccggggcag ttcacgtgcc gcacggggcggtgtatccgg aaggagctgc gctgtgatgg 1440 ctgggccgac tgcaccgacc acagcgatgagctcaactgc agttgcgacg ccggccacca 1500 gttcacgtgc aagaacaagt tctgcaagcccctcttctgg gtctgcgaca gtgtgaacga 1560 ctgcggagac aacagcgacg agcaggggtgcagttgtccg gcccagacct tcaggtgttc 1620 caatgggaag tgcctctcga aaagccagcagtgcaatggg aaggacgact gtggggacgg 1680 gtccgacgag gcctcctgcc ccaaggtgaacgtcgtcact tgtaccaaac acacctaccg 1740 ctgcctcaat gggctctgct tgagcaagggcaaccctgag tgtgacggga aggaggactg 1800 tagcgacggc tcagatgaga aggactgcgactgtgggctg cggtcattca cgagacaggc 1860 tcgtgttgtt gggggcacgg atgcggatgagggcgagtgg ccctggcagg taagcctgca 1920 tgctctgggc cagggccaca tctgcggtgcttccctcatc tctcccaact ggctggtctc 1980 tgccgcacac tgctacatcg atgacagaggattcaggtac tcagacccca cgcagtggac 2040 ggccttcctg ggcttgcacg accagagccagcgcagcgcc cctggggtgc aggagcgcag 2100 gctcaagcgc atcatctccc accccttcttcaatgacttc accttcgact atgacatcgc 2160 gctgctggag ctggagaaac cggcagagtacagctccatg gtgcggccca tctgcctgcc 2220 ggacgcctcc catgtcttcc ctgccggcaaggccatctgg gtcacgggct ggggacacac 2280 ccagtatgga ggcactggcg cgctgatcctgcaaaagggt gagatccgcg tcatcaacca 2340 gaccacctgc gagaacctcc tgccgcagcagatcacgccg cgcatgatgt gcgtgggctt 2400 cctcagcggc ggcgtggact cctgccagggtgattccggg ggacccctgt ccagcgtgga 2460 ggcggatggg cggatcttcc aggccggtgtggtgagctgg ggagacggct gcgctcagag 2520 gaacaagcca ggcgtgtaca caaggctccctctgtttcgg gactggatca aagagaacac 2580 tggggtatag gggccggggc cacccaaatgtgtacacctg cggggccacc catcgtccac 2640 cccagtgtgc acgcctgcag gctggagactggaccgctga ctgcaccagc gcccccagaa 2700 catacactgt gaactcaatc tccagggctccaaatctgcc tagaaaacct ctcgcttcct 2760 cagcctccaa agtggagctg ggaggtagaaggggaggaca ctggtggttc tactgaccca 2820 actgggggca aaggtttgaa gacacagcctcccccgccag ccccaagctg ggccgaggcg 2880 cgtttgtgta tatctgcctc ccctgtctgtaaggagcagc gggaacggag cttcggagcc 2940 tcctcagtga aggtggtggg gctgccggatctgggctgtg gggcccttgg gccacgctct 3000 tgaggaagcc caggctcgga ggaccctggaaaacagacgg gtctgagact gaaattgttt 3060 taccagctcc cagggtggac ttcagtgtgtgtatttgtgt aaatgggtaa aacaatttat 3120 ttctttttaa aaaaaaaaaa aaaaaaa 31472 855 PRT Homo sapiens TADG-15 2 Met Gly Ser Asp Arg Ala Arg Lys Gly GlyGly Gly Pro Lys Asp 5 10 15 Phe Gly Ala Gly Leu Lys Tyr Asn Ser Arg HisGlu Lys Val Asn 20 25 30 Gly Leu Glu Glu Gly Val Glu Phe Leu Pro Val AsnAsn Val Lys 35 40 45 Lys Val Glu Lys His Gly Pro Gly Arg Trp Val Val LeuAla Ala 50 55 60 Val Leu Ile Gly Leu Leu Leu Val Leu Leu Gly Ile Gly PheLeu 65 70 75 Val Trp His Leu Gln Tyr Arg Asp Val Arg Val Gln Lys Val Phe80 85 90 Asn Gly Tyr Met Arg Ile Thr Asn Glu Asn Phe Val Asp Ala Tyr 95100 105 Glu Asn Ser Asn Ser Thr Glu Phe Val Ser Leu Ala Ser Lys Val 110115 120 Lys Asp Ala Leu Lys Leu Leu Tyr Ser Gly Val Pro Phe Leu Gly 125130 135 Pro Tyr His Lys Glu Ser Ala Val Thr Ala Phe Ser Glu Gly Ser 140145 150 Val Ile Ala Tyr Tyr Trp Ser Glu Phe Ser Ile Pro Gln His Leu 155160 165 Val Glu Glu Ala Glu Arg Val Met Ala Glu Glu Arg Val Val Met 170175 180 Leu Pro Pro Arg Ala Arg Ser Leu Lys Ser Phe Val Val Thr Ser 185190 195 Val Val Ala Phe Pro Thr Asp Ser Lys Thr Val Gln Arg Thr Gln 200205 210 Asp Asn Ser Cys Ser Phe Gly Leu His Ala Arg Gly Val Glu Leu 215220 225 Met Arg Phe Thr Thr Pro Gly Phe Pro Asp Ser Pro Tyr Pro Ala 230235 240 His Ala Arg Cys Gln Trp Ala Leu Arg Gly Asp Ala Asp Ser Val 245250 255 Leu Ser Leu Thr Phe Arg Ser Phe Asp Leu Ala Ser Cys Asp Glu 260265 270 Arg Gly Ser Asp Leu Val Thr Val Tyr Asn Thr Leu Ser Pro Met 275280 285 Glu Pro His Ala Leu Val Gln Leu Cys Gly Thr Tyr Pro Pro Ser 290295 300 Tyr Asn Leu Thr Phe His Ser Ser Gln Asn Val Leu Leu Ile Thr 305310 315 Leu Ile Thr Asn Thr Glu Arg Arg His Pro Gly Phe Glu Ala Thr 320325 330 Phe Phe Gln Leu Pro Arg Met Ser Ser Cys Gly Gly Arg Leu Arg 335340 345 Lys Ala Gln Gly Thr Phe Asn Ser Pro Tyr Tyr Pro Gly His Tyr 350355 360 Pro Pro Asn Ile Asp Cys Thr Trp Asn Ile Glu Val Pro Asn Asn 365370 375 Gln His Val Lys Val Ser Phe Lys Phe Phe Tyr Leu Leu Glu Pro 380385 390 Gly Val Pro Ala Gly Thr Cys Pro Lys Asp Tyr Val Glu Ile Asn 395400 405 Gly Glu Lys Tyr Cys Gly Glu Arg Ser Gln Phe Val Val Thr Ser 410415 420 Asn Ser Asn Lys Ile Thr Val Arg Phe His Ser Asp Gln Ser Tyr 425430 435 Thr Asp Thr Gly Phe Leu Ala Glu Tyr Leu Ser Tyr Asp Ser Ser 440445 450 Asp Pro Cys Pro Gly Gln Phe Thr Cys Arg Thr Gly Arg Cys Ile 455460 465 Arg Lys Glu Leu Arg Cys Asp Gly Trp Ala Asp Cys Thr Asp His 470475 480 Ser Asp Glu Leu Asn Cys Ser Cys Asp Ala Gly His Gln Phe Thr 485490 495 Cys Lys Asn Lys Phe Cys Lys Pro Leu Phe Trp Val Cys Asp Ser 500505 510 Val Asn Asp Cys Gly Asp Asn Ser Asp Glu Gln Gly Cys Ser Cys 515520 525 Pro Ala Gln Thr Phe Arg Cys Ser Asn Gly Lys Cys Leu Ser Lys 530535 540 Ser Gln Gln Cys Asn Gly Lys Asp Asp Cys Gly Asp Gly Ser Asp 545550 555 Glu Ala Ser Cys Pro Lys Val Asn Val Val Thr Cys Thr Lys His 560565 570 Thr Tyr Arg Cys Leu Asn Gly Leu Cys Leu Ser Lys Gly Asn Pro 575580 585 Glu Cys Asp Gly Lys Glu Asp Cys Ser Asp Gly Ser Asp Glu Lys 590595 600 Asp Cys Asp Cys Gly Leu Arg Ser Phe Thr Arg Gln Ala Arg Val 605610 615 Val Gly Gly Thr Asp Ala Asp Glu Gly Glu Trp Pro Trp Gln Val 620625 630 Ser Leu His Ala Leu Gly Gln Gly His Ile Cys Gly Ala Ser Leu 635640 645 Ile Ser Pro Asn Trp Leu Val Ser Ala Ala His Cys Tyr Ile Asp 650655 660 Asp Arg Gly Phe Arg Tyr Ser Asp Pro Thr Gln Trp Thr Ala Phe 665670 675 Leu Gly Leu His Asp Gln Ser Gln Arg Ser Ala Pro Gly Val Gln 680685 690 Glu Arg Arg Leu Lys Arg Ile Ile Ser His Pro Phe Phe Asn Asp 695700 705 Phe Thr Phe Asp Tyr Asp Ile Ala Leu Leu Glu Leu Glu Lys Pro 710715 720 Ala Glu Tyr Ser Ser Met Val Arg Pro Ile Cys Leu Pro Asp Ala 725730 735 Ser His Val Phe Pro Ala Gly Lys Ala Ile Trp Val Thr Gly Trp 740745 750 Gly His Thr Gln Tyr Gly Gly Thr Gly Ala Leu Ile Leu Gln Lys 755760 765 Gly Glu Ile Arg Val Ile Asn Gln Thr Thr Cys Glu Asn Leu Leu 770775 780 Pro Gln Gln Ile Thr Pro Arg Met Met Cys Val Gly Phe Leu Ser 785790 795 Gly Gly Val Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Ser 800805 810 Ser Val Glu Ala Asp Gly Arg Ile Phe Gln Ala Gly Val Val Ser 815820 825 Trp Gly Asp Gly Cys Ala Gln Arg Asn Lys Pro Gly Val Tyr Thr 830835 840 Arg Leu Pro Leu Phe Arg Asp Trp Ile Lys Glu Asn Thr Gly Val 845850 855 3 256 PRT Homo sapiens Hepsin 3 Arg Ile Val Gly Gly Arg Asp ThrSer Leu Gly Arg Trp Pro Trp 5 10 15 Gln Val Ser Leu Arg Tyr Asp Gly AlaHis Leu Cys Gly Gly Ser 20 25 30 Leu Leu Ser Gly Asp Trp Val Leu Thr AlaAla His Cys Phe Pro 35 40 45 Glu Arg Asn Arg Val Leu Ser Arg Trp Arg ValPhe Ala Gly Ala 50 55 60 Val Ala Gln Ala Ser Pro His Gly Leu Gln Leu GlyVal Gln Ala 65 70 75 Val Val Tyr His Gly Gly Tyr Leu Pro Phe Arg Asp ProAsn Ser 80 85 90 Glu Glu Asn Ser Asn Asp Ile Ala Leu Val His Leu Ser SerPro 95 100 105 Leu Pro Leu Thr Glu Tyr Ile Gln Pro Val Cys Leu Pro AlaAla 110 115 120 Gly Gln Ala Leu Val Asp Gly Lys Ile Cys Thr Val Thr GlyTrp 125 130 135 Gly Asn Thr Gln Tyr Tyr Gly Gln Gln Ala Gly Val Leu GlnGlu 140 145 150 Ala Arg Val Pro Ile Ile Ser Asn Asp Val Cys Asn Gly AlaAsp 155 160 165 Phe Tyr Gly Asn Gln Ile Lys Pro Lys Met Phe Cys Ala GlyTyr 170 175 180 Pro Glu Gly Gly Ile Asp Ala Cys Gln Gly Asp Ser Gly GlyPro 185 190 195 Phe Val Cys Glu Asp Ser Ile Ser Arg Thr Pro Arg Trp ArgLeu 200 205 210 Cys Gly Ile Val Ser Trp Gly Thr Gly Cys Ala Leu Ala GlnLys 215 220 225 Pro Gly Val Tyr Thr Lys Val Ser Asp Phe Arg Glu Trp IlePhe 230 235 240 Gln Ala Ile Lys Thr His Ser Glu Ala Ser Gly Met Val ThrGln 245 250 255 Leu 4 225 PRT Homo sapiens SCCE 4 Lys Ile Ile Asp GlyAla Pro Cys Ala Arg Gly Ser His Pro Trp 5 10 15 Gln Val Ala Leu Leu SerGly Asn Gln Leu His Cys Gly Gly Val 20 25 30 Leu Val Asn Glu Arg Trp ValLeu Thr Ala Ala His Cys Lys Met 35 40 45 Asn Glu Tyr Thr Val His Leu GlySer Asp Thr Leu Gly Asp Arg 50 55 60 Arg Ala Gln Arg Ile Lys Ala Ser LysSer Phe Arg His Pro Gly 65 70 75 Tyr Ser Thr Gln Thr His Val Asn Asp LeuMet Leu Val Lys Leu 80 85 90 Asn Ser Gln Ala Arg Leu Ser Ser Met Val LysLys Val Arg Leu 95 100 105 Pro Ser Arg Cys Glu Pro Pro Gly Thr Thr CysThr Val Ser Gly 110 115 120 Trp Gly Thr Thr Thr Ser Pro Asp Val Thr PhePro Ser Asp Leu 125 130 135 Met Cys Val Asp Val Lys Leu Ile Ser Pro GlnAsp Cys Thr Lys 140 145 150 Val Tyr Lys Asp Leu Leu Glu Asn Ser Met LeuCys Ala Gly Ile 155 160 165 Pro Asp Ser Lys Lys Asn Ala Cys Asn Gly AspSer Gly Gly Pro 170 175 180 Leu Val Cys Arg Gly Thr Leu Gln Gly Leu ValSer Trp Gly Thr 185 190 195 Phe Pro Cys Gly Gln Pro Asn Asp Pro Gly ValTyr Thr Gln Val 200 205 210 Cys Lys Phe Thr Lys Trp Ile Asn Asp Thr MetLys Lys His Arg 215 220 225 5 225 PRT Homo sapiens Trypsin 5 Lys Ile ValGly Gly Tyr Asn Cys Glu Glu Asn Ser Val Pro Tyr 5 10 15 Gln Val Ser LeuAsn Ser Gly Tyr His Phe Cys Gly Gly Ser Leu 20 25 30 Ile Asn Glu Gln TrpVal Val Ser Ala Gly His Cys Tyr Lys Ser 35 40 45 Arg Ile Gln Val Arg LeuGly Glu His Asn Ile Glu Val Leu Glu 50 55 60 Gly Asn Glu Gln Phe Ile AsnAla Ala Lys Ile Ile Arg His Pro 65 70 75 Gln Tyr Asp Arg Lys Thr Leu AsnAsn Asp Ile Met Leu Ile Lys 80 85 90 Leu Ser Ser Arg Ala Val Ile Asn AlaArg Val Ser Thr Ile Ser 95 100 105 Leu Pro Thr Ala Pro Pro Ala Thr GlyThr Lys Cys Leu Ile Ser 110 115 120 Gly Trp Gly Asn Thr Ala Ser Ser GlyAla Asp Tyr Pro Asp Glu 125 130 135 Leu Gln Cys Leu Asp Ala Pro Val LeuSer Gln Ala Lys Cys Glu 140 145 150 Ala Ser Tyr Pro Gly Lys Ile Thr SerAsn Met Phe Cys Val Gly 155 160 165 Phe Leu Glu Gly Gly Lys Asp Ser CysGln Gly Asp Ser Gly Gly 170 175 180 Pro Val Val Cys Asn Gly Gln Leu GlnGly Val Val Ser Trp Gly 185 190 195 Asp Gly Cys Ala Gln Lys Asn Lys ProGly Val Tyr Thr Lys Val 200 205 210 Tyr Asn Tyr Val Lys Trp Ile Lys AsnThr Ile Ala Ala Asn Ser 215 220 225 6 231 PRT Homo sapiens Chymotrypsin6 Arg Ile Val Asn Gly Glu Asp Ala Val Pro Gly Ser Trp Pro Trp 5 10 15Gln Val Ser Leu Gln Asp Lys Thr Gly Phe His Phe Cys Gly Gly 20 25 30 SerLeu Ile Ser Glu Asp Trp Val Val Thr Ala Ala His Cys Gly 35 40 45 Val ArgThr Ser Asp Val Val Val Ala Gly Glu Phe Asp Gln Gly 50 55 60 Ser Asp GluGlu Asn Ile Gln Val Leu Lys Ile Ala Lys Val Phe 65 70 75 Lys Asn Pro LysPhe Ser Ile Leu Thr Val Asn Asn Asp Ile Thr 80 85 90 Leu Leu Lys Leu AlaThr Pro Ala Arg Phe Ser Gln Thr Val Ser 95 100 105 Ala Val Cys Leu ProSer Ala Asp Asp Asp Phe Pro Ala Gly Thr 110 115 120 Leu Cys Ala Thr ThrGly Trp Gly Lys Thr Lys Tyr Asn Ala Asn 125 130 135 Lys Thr Pro Asp LysLeu Gln Gln Ala Ala Leu Pro Leu Leu Ser 140 145 150 Asn Ala Glu Cys LysLys Ser Trp Gly Arg Arg Ile Thr Asp Val 155 160 165 Met Ile Cys Ala GlyAla Ser Gly Val Ser Ser Cys Met Gly Asp 170 175 180 Ser Gly Gly Pro LeuVal Cys Gln Lys Asp Gly Ala Trp Thr Leu 185 190 195 Val Gly Ile Val SerTrp Gly Ser Asp Thr Cys Ser Thr Ser Ser 200 205 210 Pro Gly Val Tyr AlaArg Val Thr Lys Leu Ile Pro Trp Val Gln 215 220 225 Lys Ile Leu Ala AlaAsn 230 7 255 PRT Homo sapiens Factor 7 7 Arg Ile Val Gly Gly Lys ValCys Pro Lys Gly Glu Cys Pro Trp 5 10 15 Gln Val Leu Leu Leu Val Asn GlyAla Gln Leu Cys Gly Gly Thr 20 25 30 Leu Ile Asn Thr Ile Trp Val Val SerAla Ala His Cys Phe Asp 35 40 45 Lys Ile Lys Asn Trp Arg Asn Leu Ile AlaVal Leu Gly Glu His 50 55 60 Asp Leu Ser Glu His Asp Gly Asp Glu Gln SerArg Arg Val Ala 65 70 75 Gln Val Ile Ile Pro Ser Thr Tyr Val Pro Gly ThrThr Asn His 80 85 90 Asp Ile Ala Leu Leu Arg Leu His Gln Pro Val Val LeuThr Asp 95 100 105 His Val Val Pro Leu Cys Leu Pro Glu Arg Thr Phe SerGlu Arg 110 115 120 Thr Leu Ala Phe Val Arg Phe Ser Leu Val Ser Gly TrpGly Gln 125 130 135 Leu Leu Asp Arg Gly Ala Thr Ala Leu Glu Leu Met ValLeu Asn 140 145 150 Val Pro Arg Leu Met Thr Gln Asp Cys Leu Gln Gln SerArg Lys 155 160 165 Val Gly Asp Ser Pro Asn Ile Thr Glu Tyr Met Phe CysAla Gly 170 175 180 Tyr Ser Asp Gly Ser Lys Asp Ser Cys Lys Gly Asp SerGly Gly 185 190 195 Pro His Ala Thr His Tyr Arg Gly Thr Trp Tyr Leu ThrGly Ile 200 205 210 Val Ser Trp Gly Gln Gly Cys Ala Thr Val Gly His PheGly Val 215 220 225 Tyr Thr Arg Val Ser Gln Tyr Ile Glu Trp Leu Gln LysLeu Met 230 235 240 Arg Ser Glu Pro Arg Pro Gly Val Leu Leu Arg Ala ProPhe Pro 245 250 255 8 253 PRT Homo sapiens Tissue plasminogen activator8 Arg Ile Lys Gly Gly Leu Phe Ala Asp Ile Ala Ser His Pro Trp 5 10 15Gln Ala Ala Ile Phe Ala Lys His Arg Arg Ser Pro Gly Glu Arg 20 25 30 PheLeu Cys Gly Gly Ile Leu Ile Ser Ser Cys Trp Ile Leu Ser 35 40 45 Ala AlaHis Cys Phe Gln Glu Arg Phe Pro Pro His His Leu Thr 50 55 60 Val Ile LeuGly Arg Thr Tyr Arg Val Val Pro Gly Glu Glu Glu 65 70 75 Gln Lys Phe GluVal Glu Lys Tyr Ile Val His Lys Glu Phe Asp 80 85 90 Asp Asp Thr Tyr AspAsn Asp Ile Ala Leu Leu Gln Leu Lys Ser 95 100 105 Asp Ser Ser Arg CysAla Gln Glu Ser Ser Val Val Arg Thr Val 110 115 120 Cys Leu Pro Pro AlaAsp Leu Gln Leu Pro Asp Trp Thr Glu Cys 125 130 135 Glu Leu Ser Gly TyrGly Lys His Glu Ala Leu Ser Pro Phe Tyr 140 145 150 Ser Glu Arg Leu LysGlu Ala His Val Arg Leu Tyr Pro Ser Ser 155 160 165 Arg Cys Thr Ser GlnHis Leu Leu Asn Arg Thr Val Thr Asp Asn 170 175 180 Met Leu Cys Ala GlyAsp Thr Arg Ser Gly Gly Pro Gln Ala Asn 185 190 195 Leu His Asp Ala CysGln Gly Asp Ser Gly Gly Pro Leu Val Cys 200 205 210 Leu Asn Asp Gly ArgMet Thr Leu Val Gly Ile Ile Ser Trp Gly 215 220 225 Leu Gly Cys Gly GlnLys Asp Val Pro Gly Val Tyr Thr Lys Val 230 235 240 Thr Asn Tyr Leu AspTrp Ile Arg Asp Asn Met Arg Pro 245 250 9 2900 DNA Homo sapiens SNC-19;GeneBank Accession No. #U20428 9 cgctgggtgg tgctggcagc cgtgctgatcggcctcctct tggtcttgct ggggatcggc 60 ttcctggtgt ggcatttgca gtaccgggacgtgcgtgtcc agaaggtctt caatggctac 120 atgaggatca caaatgagaa ttttgtggatgcctacgaga actccaactc cactgagttt 180 gtaagcctgg ccagcaaggt gaaggacgcgctgaagctgc tgtacagcgg agtcccattc 240 ctgggcccct accacaagga gtcggctgtgacggccttca gcgagggcag cgtcatcgcc 300 tactactggt ctgagttcag catcccgcagcacctggttg aggaggccga gcgcgtcatg 360 gccaggagcg cgtagtcatg ctgcccccgcgggcgcgctc cctgaagtcc tttgtggtca 420 cctcagtggt ggctttcccc acggactccaaaacagtaca gaggacccag gacaacagct 480 gcagctttgg cctgcacgcc gcggtgtggagctgatgcgc ttcaccacgc cggcttccct 540 gacagcccct accccgctca tgcccgctgccagtgggctg cggggacgcg acgcagtgct 600 gagctactcg agctgactcg cagcttgactgcgcctcgac gagcgcggca gcgacctggt 660 gacgtgtaca acaccctgag ccccatggagccccacgcct ggtgagtgtg tggcacctac 720 cctccctcct acaacctgac cttccactccctcccacgaa cgtcctgctc atcacactga 780 taaccaacac tgacgcggca tcccggctttgaggccacct tcttccagct gcctaggatg 840 agcagctgtg gaggccgctt acgtaaagcccaggggacat tcaacagccc ctactaccca 900 ggccactacc cacccaacat tgactgcacatggaaaattg aggtgcccaa caaccagcat 960 gtgaaggtgc gcttcaaatt cttctacctgctggagcccg gcgtgcctgc gggcacctgc 1020 cccaaggact acgtggagat caatggggagaaatactgcg gagagaggtc ccagttcgtc 1080 gtcaccagca acagcaacaa gatcacagttcgcttccact cagatcagtc ctacaccgac 1140 accggcttct tagctgaata cctctcctacgactccagtg acccatgccc ggggcagttc 1200 acgtgccgca cggggcggtg tatccggaaggagctgcgct gtgatggctg ggcgactgca 1260 ccgaccacag cgatgagctc aactgcagttgcgacgccgg ccaccagttc acgtgcaaga 1320 gcaagttctg caagctcttc tgggtctgcgacagtgtgaa cgagtgcgga gacaacagcg 1380 acgagcaggg ttgcatttgt ccggacccagaccttcaggt gttccaatgg gaagtgcctc 1440 tcgaaaagcc agcagtgcaa tgggaaggacgactgtgggg acgggtccga cgaggcctcc 1500 tgccccaagg tgaacgtcgt cacttgtaccaaacacacct accgctgcct caatgggctc 1560 tgcttgagca agggcaaccc tgagtgtgacgggaaggagg actgtagcga cggctcagat 1620 gagaaggact gcgactgtgg gctgcggtcattcacgagac aggctcgtgt tgttgggggc 1680 acggatgcgg atgagggcga gtggccctggcaggtaagcc tgcatgctct gggccagggc 1740 cacatctgcg gtgcttccct catctctcccaactggctgg tctctgccgc acactgctac 1800 atcgatgaca gaggattcag gtactcagaccccacgcagg acggccttcc tgggcttgca 1860 cgaccagagc cagcgcaggc cctggggtgcaggagcgcag gctcaagcgc atcatctccc 1920 accccttctt caatgacttc accttcgactatgacatcgc gctgctggag ctggagaaac 1980 cggcagagta cagctccatg gtgcggcccatctgcctgcc ggacgcctgc catgtcttcc 2040 ctgccggcaa ggccatctgg gtcacgggctggggacacac ccagtatgga ggcactggcg 2100 cgctgatcct gcaaaagggt gagatccgcgtcatcaacca gaccacctgc gagaacctcc 2160 tgccgcagca gatcacgccg cgcatgatgtgcgtgggctt cctcagcggc ggcgtggact 2220 cctgccaggg tgattccggg ggacccctgtccagcgtgga ggcggatggg cggatcttcc 2280 aggccggtgt ggtgagctgg ggagacgctgcgctcagagg aacaagccag gcgtgtacac 2340 aaggctccct ctgtttcggg aatggatcaaagagaacact ggggtatagg ggccggggcc 2400 acccaaatgt gtacacctgc ggggccacccatcgtccacc ccagtgtgca cgcctgcagg 2460 ctggagactc gcgcaccgtg acctgcaccagcgccccaga acatacactg tgaactcatc 2520 tccaggctca aatctgctag aaaacctctcgcttcctcag cctccaaagt ggagctggga 2580 gggtagaagg ggaggaacac tggtggttctactgacccaa ctggggcaag gtttgaagca 2640 cagctccggc agcccaagtg ggcgaggacgcgtttgtgca tactgccctg ctctatacac 2700 ggaagacctg gatctctagt gagtgtgactgccggatctg gctgtggtcc ttggccacgc 2760 ttcttgagga agcccaggct cggaggaccctggaaaacag acgggtctga gactgaaaat 2820 ggtttaccag ctcccaggtg acttcagtgtgtgtattgtg taaatgagta aaacatttta 2880 tttcttttta aaaaaaaaaa 2900 10 902PRT Mus musculus Epithin 10 Met Gly Ser Asn Arg Gly Arg Lys Ala Gly GlyGly Ser Gln Asp 5 10 15 Phe Gly Ala Gly Leu Lys Tyr Asp Ser Arg Leu GluAsn Met Asn 20 25 30 Gly Phe Glu Glu Gly Val Glu Phe Leu Pro Ala Asn AsnAla Lys 35 40 45 Lys Val Glu Lys Arg Gly Pro Arg Arg Trp Val Val Leu ValAla 50 55 60 Val Leu Phe Ser Phe Leu Leu Leu Ser Leu Met Ala Gly Leu Leu65 70 75 Val Trp His Phe His Tyr Arg Asn Val Arg Val Gln Lys Val Phe 8085 90 Asn Gly His Leu Arg Ile Thr Asn Glu Ile Phe Leu Asp Ala Tyr 95 100105 Glu Asn Ser Thr Ser Thr Glu Phe Ile Ser Leu Ala Ser Gln Val 110 115120 Lys Glu Ala Leu Lys Leu Leu Tyr Asn Glu Val Pro Val Leu Gly 125 130135 Pro Tyr His Lys Lys Ser Ala Val Thr Ala Phe Ser Glu Gly Ser 140 145150 Val Ile Ala Tyr Tyr Trp Ser Glu Phe Ser Ile Pro Pro His Leu 155 160165 Ala Glu Glu Val Asp Arg Ala Met Ala Val Glu Arg Val Val Thr 170 175180 Leu Pro Pro Arg Ala Arg Ala Leu Lys Ser Phe Val Leu Thr Ser 185 190195 Val Val Ala Phe Pro Ile Asp Pro Arg Met Leu Gln Arg Thr Gln 200 205210 Asp Asn Ser Cys Ser Phe Ala Leu His Ala His Gly Ala Ala Val 215 220225 Thr Arg Phe Thr Thr Pro Gly Phe Pro Asn Ser Pro Tyr Pro Ala 230 235240 His Ala Arg Cys Gln Trp Val Leu Arg Gly Asp Ala Asp Ser Val 245 250255 Leu Ser Leu Thr Phe Arg Ser Phe Asp Val Ala Pro Cys Asp Glu 260 265270 His Gly Ser Asp Leu Val Thr Val Tyr Asp Ser Leu Ser Pro Met 275 280285 Glu Pro His Ala Val Val Arg Leu Cys Gly Thr Phe Ser Pro Ser 290 295300 Tyr Asn Leu Thr Phe Leu Ser Ser Gln Asn Val Phe Leu Val Thr 305 310315 Leu Ile Thr Asn Thr Gly Arg Arg His Leu Gly Phe Glu Ala Thr 320 325330 Phe Phe Gln Leu Pro Lys Met Ser Ser Cys Gly Gly Val Leu Ser 335 340345 Asp Thr Gln Gly Thr Phe Ser Ser Pro Tyr Tyr Pro Gly His Tyr 350 355360 Pro Pro Asn Ile Asn Cys Thr Trp Asn Ile Lys Val Pro Asn Asn 365 370375 Arg Asn Val Lys Val Arg Phe Lys Leu Phe Tyr Leu Val Asp Pro 380 385390 Asn Val Pro Val Gly Ser Cys Thr Lys Asp Tyr Val Glu Ile Asn 395 400405 Gly Glu Lys Gly Ser Gly Glu Arg Ser Gln Phe Val Val Ser Ser 410 415420 Asn Ser Ser Lys Ile Thr Val His Phe His Ser Asp His Ser Tyr 425 430435 Thr Asp Thr Gly Phe Leu Ala Glu Tyr Leu Ser Tyr Asp Ser Asn 440 445450 Asp Pro Cys Pro Gly Met Phe Met Cys Lys Thr Gly Arg Cys Ile 455 460465 Arg Lys Glu Leu Arg Cys Asp Gly Trp Ala Asp Cys Pro Asp Tyr 470 475480 Ser Asp Glu Arg Tyr Cys Arg Cys Asn Ala Thr His Gln Phe Thr 485 490495 Cys Lys Asn Gln Phe Cys Lys Pro Leu Phe Trp Val Cys Asp Ser 500 505510 Val Asn Asp Cys Gly Asp Gly Ser Asp Glu Glu Gly Cys Ser Cys 515 520525 Pro Ala Gly Ser Phe Lys Cys Ser Asn Gly Lys Cys Leu Pro Gln 530 535540 Ser Gln Lys Cys Asn Gly Lys Asp Asn Cys Gly Asp Gly Ser Asp 545 550555 Glu Ala Ser Cys Asp Ser Val Asn Val Val Ser Cys Thr Lys Tyr 560 565570 Thr Tyr Arg Cys Gln Asn Gly Leu Cys Leu Ser Lys Gly Asn Pro 575 580585 Glu Cys Asp Gly Lys Thr Asp Cys Ser Asp Gly Ser Asp Glu Lys 590 595600 Asn Cys Asp Cys Gly Leu Arg Ser Phe Thr Lys Gln Ala Arg Val 605 610615 Val Gly Gly Thr Asn Ala Asp Glu Gly Glu Trp Pro Trp Gln Val 620 625630 Ser Leu His Ala Leu Gly Gln Gly His Leu Cys Gly Ala Ser Leu 635 640645 Ile Ser Pro Asp Trp Leu Val Ser Ala Ala His Cys Phe Gln Asp 650 655660 Asp Lys Asn Phe Lys Tyr Ser Asp Tyr Thr Met Trp Thr Ala Phe 665 670675 Leu Gly Leu Leu Asp Gln Ser Lys Arg Ser Ala Ser Gly Val Gln 680 685690 Glu Leu Lys Leu Lys Arg Ile Ile Thr His Pro Ser Phe Asn Asp 695 700705 Phe Thr Phe Asp Tyr Asp Ile Ala Leu Leu Glu Leu Glu Lys Ser 710 715720 Val Glu Tyr Ser Thr Val Val Arg Pro Ile Cys Leu Pro Asp Ala 725 730735 Thr His Val Phe Pro Ala Gly Lys Ala Ile Trp Val Thr Gly Trp 740 745750 Gly His Thr Lys Glu Gly Gly Thr Gly Ala Leu Ile Leu Gln Lys 755 760765 Gly Glu Ile Arg Val Ile Asn Gln Thr Thr Cys Glu Asp Leu Met 770 775780 Pro Gln Gln Ile Thr Pro Arg Met Met Cys Val Gly Phe Leu Ser 785 790795 Gly Gly Val Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Ser 800 805810 Ser Ala Glu Lys Asp Gly Arg Met Phe Gln Ala Gly Val Val Ser 815 820825 Trp Gly Glu Gly Cys Ala Gln Arg Asn Lys Pro Gly Val Tyr Thr 830 835840 Arg Leu Pro Cys Ser Ser Gly Leu Asp Gln Arg Ala His Trp Gly 845 850855 Ile Ala Ala Trp Thr Asp Ser Arg Pro Gln Thr Pro Thr Gly Met 860 865870 Pro Asp Met His Thr Trp Ile Gln Glu Arg Asn Thr Asp Asp Ile 875 880885 Tyr Ala Val Ala Ser Pro Pro Gln His Asn Pro Asp Cys Glu Leu 890 895900 His Pro 11 23 DNA Artificial sequence n=Inosine 6, 9, 12, 15, 18Degenerate oligonucleotide primer 11 tgggtngtna cngcngcnca ytg 23 12 20DNA Artificial sequence n=Inosine 3, 6, 9, 12, 18 Degenerateoligonucleotide primer 12 arnggnccnc cnswrtcncc 20 13 12 PRT Homosapiens Fragment of TADG-15 13 Leu Phe Arg Asp Trp Ile Lys Glu Asn ThrGly Val 5 10 14 20 DNA Artificial sequence TADG-15 forwardoligonucleotide primer 14 atgacagagg attcaggtac 20 15 20 DNA Artificialsequence TADG-15 reverse oligonucleotide primer 15 gaaggtgaag tcattgaaga20 16 20 DNA Artificial sequence (-tubulin forward oligonucleotideprimer 16 cgcatcaacg tgtactacaa 20 17 20 DNA Artificial sequence(-tubulin reverse oligonucleotide primer 17 tacgagctgg tggactgaga 20 183147 RNA Artificial sequence Antisense of TADG-15 18 uuuuuuuuuuuuuuuuuuua aaaagaaaua aauuguuuua cccauuuaca 50 caaauacaca cacugaaguccacccuggga gcugguaaaa caauuucagu 100 cucagacccg ucuguuuucc aggguccuccgagccugggc uuccucaaga 150 gcguggccca agggccccac agcccagauc cggcagccccaccaccuuca 200 cugaggaggc uccgaagcuc cguucccgcu gcuccuuaca gacaggggag250 gcagauauac acaaacgcgc cucggcccag cuuggggcug gcgggggagg 300cugugucuuc aaaccuuugc ccccaguugg gucaguagaa ccaccagugu 350 ccuccccuucuaccucccag cuccacuuug gaggcugagg aagcgagagg 400 uuuucuaggc agauuuggagcccuggagau ugaguucaca guguauguuc 450 ugggggcgcu ggugcaguca gcgguccagucuccagccug caggcgugca 500 cacuggggug gacgaugggu ggccccgcag guguacacauuuggguggcc 550 ccggccccua uaccccagug uucucuuuga uccagucccg aaacagaggg600 agccuugugu acacgccugg cuuguuccuc ugagcgcagc cgucucccca 650gcucaccaca ccggccugga agauccgccc auccgccucc acgcuggaca 700 ggggucccccggaaucaccc uggcaggagu ccacgccgcc gcugaggaag 750 cccacgcaca ucaugcgcggcgugaucugc ugcggcagga gguucucgca 800 gguggucugg uugaugacgc ggaucucacccuuuugcagg aucagcgcgc 850 cagugccucc auacugggug uguccccagc ccgugacccagauggccuug 900 ccggcaggga agacauggga ggcguccggc aggcagaugg gccgcaccau950 ggagcuguac ucugccgguu ucuccagcuc cagcagcgcg augucauagu 1000cgaaggugaa gucauugaag aagggguggg agaugaugcg cuugagccug 1050 cgcuccugcaccccaggggc gcugcgcugg cucuggucgu gcaagcccag 1100 gaaggccguc cacugcguggggucugagua ccugaauccu cugucaucga 1150 uguagcagug ugcggcagag accagccaguugggagagau gagggaagca 1200 ccgcagaugu ggcccuggcc cagagcaugc aggcuuaccugccagggcca 1250 cucgcccuca uccgcauccg ugcccccaac aacacgagcc ugucucguga1300 augaccgcag cccacagucg caguccuucu caucugagcc gucgcuacag 1350uccuccuucc cgucacacuc aggguugccc uugcucaagc agagcccauu 1400 gaggcagcgguagguguguu ugguacaagu gacgacguuc accuuggggc 1450 aggaggccuc gucggacccguccccacagu cguccuuccc auugcacugc 1500 uggcuuuucg agaggcacuu cccauuggaacaccugaagg ucugggccgg 1550 acaacugcac cccugcucgu cgcuguuguc uccgcagucguucacacugu 1600 cgcagaccca gaagaggggc uugcagaacu uguucuugca cgugaacugg1650 uggccggcgu cgcaacugca guugagcuca ucgcuguggu cggugcaguc 1700ggcccagcca ucacagcgca gcuccuuccg gauacaccgc cccgugcggc 1750 acgugaacugccccgggcau gggucacugg agucguagga gagguauuca 1800 gcuaagaagc cggugucgguguaggacuga ucugagugga agcgaacugu 1850 gaucuuguug cuguugcugg ugacgacgaacugggaccuc ucuccgcagu 1900 auuucucccc auugaucucc acguaguccu uggggcaggugcccgcaggc 1950 acgccgggcu ccagcaggua gaagaauuug aagcucaccu ucacaugcug2000 guuguugggc accucaaugu uccaugugca gucaauguug gguggguagu 2050ggccugggua guaggggcug uugaaugucc ccugggcuuu acguaagcgg 2100 ccuccacagcugcucauccu aggcagcugg aagaaggugg ccucaaagcc 2150 gggaugccgc cgcucaguguugguuaucag ugugaugagc aggacguucu 2200 gggaggagug gaaggucagg uuguaggagggaggguaggu gccacacaac 2250 ugcaccaggg cguggggcuc cauggggcuc aggguguuguacaccgucac 2300 caggucgcug ccgcgcucgu cgcaggacgc aaggucaaag cugcggaagg2350 ugaggcucag cacugagucg gcgucccccc gcagggccca cuggcagcgg 2400gcaugagcgg gguaggggcu gucagggaag ccgggcgugg ugaagcgcau 2450 cagcuccacaccgcgggcgu gcaggccaaa gcugcagcug uuguccuggg 2500 uccucuguac uguuuuggaguccgugggga aagccaccac ugaggugacc 2550 acaaaggacu ucagggagcg cgcccgcgggggcagcauga cuacgcgcuc 2600 cucggccaug acgcgcucgg ccuccuccac caggugcugcgggaugcuga 2650 acucagacca guaguaggcg augacgcugc ccucgcugaa ggccgucaca2700 gccgacuccu ugugguaggg gcccaggaau gggacuccgc uguacagcag 2750cuucagcgcg uccuucaccu ugcuggccag gcuuacaaac ucaguggagu 2800 uggaguucucguaggcaucc acaaaauucu cauuugugau ccucauguag 2850 ccauugaaga ccuucuggacacgcacgucc cgguacugca aaugccacac 2900 caggaagccg auccccagca agaccaagaggaggccgauc agcacggcug 2950 ccagcaccac ccagcgcccc gggccaugcu uuuccaccuucuugacguug 3000 uugacuggca ggaacuccac gccuuccucc aagccauuca cuuucucgug3050 ccgggaguug uacuugaguc ccgcgccgaa guccuucggg cccccuccgc 3100ccuugcgggc ccgaucgcuc cccaugguac cccgaggccg cucuuga 3147 19 9 PRT Homosapiens Residues 68-76 of the TADG-15 protein 19 Val Leu Leu Gly Ile GlyPhe Leu Val 5 20 9 PRT Homo sapiens Residues 126-134 of the TADG-15protein 20 Leu Leu Tyr Ser Gly Val Pro Phe Leu 5 21 9 PRT Homo sapiensResidues 644-652 of the TADG-15 protein 21 Ser Leu Ile Ser Pro Asn TrpLeu Val 5 22 9 PRT Homo sapiens Residues 379-387 of the TADG-15 protein22 Lys Val Ser Phe Lys Phe Phe Tyr Leu 5 23 9 PRT Homo sapiens Residues386-394 of the TADG-15 protein 23 Tyr Leu Leu Glu Pro Gly Val Pro Ala 524 9 PRT Homo sapiens Residues 257-265 of the TADG-15 protein 24 Ser LeuThr Phe Arg Ser Phe Asp Leu 5 25 9 PRT Homo sapiens Residues 762-770 ofthe TADG-15 protein 25 Ile Leu Gln Lys Gly Glu Ile Arg Val 5 26 9 PRTHomo sapiens Residues 841-849 of the TADG-15 protein 26 Arg Leu Pro LeuPhe Arg Asp Trp Ile 5 27 9 PRT Homo sapiens Residues 64-72 of theTADG-15 protein 27 Gly Leu Leu Leu Val Leu Leu Gly Ile 5 28 9 PRT Homosapiens Residues 57-65 of the TADG-15 protein 28 Val Leu Ala Ala Val LeuIle Gly Leu 5 29 9 PRT Homo sapiens Residues 67-75 of the TADG-15protein 29 Leu Val Leu Leu Gly Ile Gly Phe Leu 5 30 9 PRT Homo sapiensResidues 379-387 of the TADG-15 protein 30 Lys Val Ser Phe Lys Phe PheTyr Leu 5 31 9 PRT Homo sapiens Residues 126-134 of the TADG-15 protein31 Leu Leu Tyr Ser Gly Val Pro Phe Leu 5 32 9 PRT Homo sapiens Residues88-96 of the TADG-15 protein 32 Lys Val Phe Asn Gly Tyr Met Arg Ile 5 339 PRT Homo sapiens Residues 670-678 of the TADG-15 protein 33 Thr GlnTrp Thr Ala Phe Leu Gly Leu 5 34 9 PRT Homo sapiens Residues 119-127 ofthe TADG-15 protein 34 Lys Val Lys Asp Ala Leu Lys Leu Leu 5 35 9 PRTHomo sapiens Residues 60-68 of the TADG-15 protein 35 Ala Val Leu IleGly Leu Leu Leu Val 5 36 9 PRT Homo sapiens Residues 62-70 of theTADG-15 protein 36 Leu Ile Gly Leu Leu Leu Val Leu Leu 5 37 9 PRT Homosapiens Residues 57-65 of the TADG-15 protein 37 Val Leu Ala Ala Val LeuIle Gly Leu 5 38 9 PRT Homo sapiens Residues 61-69 of the TADG-15protein 38 Val Leu Ile Gly Leu Leu Leu Val Leu 5 39 9 PRT Homo sapiensResidues 146-154 of the TADG-15 protein 39 Phe Ser Glu Gly Ser Val IleAla Tyr 5 40 9 PRT Homo sapiens Residues 658-666 of the TADG-15 protein40 Tyr Ile Asp Asp Arg Gly Phe Arg Tyr 5 41 9 PRT Homo sapiens Residues449-457 of the TADG-15 protein 41 Ser Ser Asp Pro Cys Pro Gly Gln Phe 542 9 PRT Homo sapiens Residues 401-409 of the TADG-15 protein 42 Tyr ValGlu Ile Asn Gly Glu Lys Tyr 5 43 9 PRT Homo sapiens Residues 387-395 ofthe TADG-15 protein 43 Leu Leu Glu Pro Gly Val Pro Ala Gly 5 44 9 PRTHomo sapiens Residues 553-561 of the TADG-15 protein 44 Gly Ser Asp GluAla Ser Cys Pro Lys 5 45 9 PRT Homo sapiens Residues 97-105 of theTADG-15 protein 45 Thr Asn Glu Asn Phe Val Asp Ala Tyr 5 46 9 PRT Homosapiens Residues 110-118 of the TADG-15 protein 46 Ser Thr Glu Phe ValSer Leu Ala Ser 5 47 9 PRT Homo sapiens Residues 811-819 of the TADG-15protein 47 Ser Val Glu Ala Asp Gly Arg Ile Phe 5 48 9 PRT Homo sapiensResidues 666-674 of the TADG-15 protein 48 Tyr Ser Asp Pro Thr Gln TrpThr Ala 5 49 9 PRT Homo sapiens Residues 709-717 of the TADG-15 protein49 Asp Tyr Asp Ile Ala Leu Leu Glu Leu 5 50 9 PRT Homo sapiens Residues408-416 of the TADG-15 protein 50 Lys Tyr Cys Gly Glu Arg Ser Gln Phe 551 9 PRT Homo sapiens Residues 754-762 of the TADG-15 protein 51 Gln TyrGly Gly Thr Gly Ala Leu Ile 5 52 9 PRT Homo sapiens Residues 153-161 ofthe TADG-15 protein 52 Ala Tyr Tyr Trp Ser Glu Phe Ser Ile 5 53 9 PRTHomo sapiens Residues 722-730 of the TADG-15 protein 53 Glu Tyr Ser SerMet Val Arg Pro Ile 5 54 9 PRT Homo sapiens Residues 326-334 of theTADG-15 protein 54 Gly Phe Glu Ala Thr Phe Phe Gln Leu 5 55 9 PRT Homosapiens Residues 304-312 of the TADG-15 protein 55 Thr Phe His Ser SerGln Asn Val Leu 5 56 9 PRT Homo sapiens Residues 707-715 of the TADG-15protein 56 Thr Phe Asp Tyr Asp Ile Ala Leu Leu 5 57 9 PRT Homo sapiensResidues 21-29 of the TADG-15 protein 57 Lys Tyr Asn Ser Arg His Glu LysVal 5 58 9 PRT Homo sapiens Residues 665-673 of the TADG-15 protein 58Arg Tyr Ser Asp Pro Thr Gln Trp Thr 5 59 9 PRT Homo sapiens Residues686-694 of the TADG-15 protein 59 Ala Pro Gly Val Gln Glu Arg Arg Leu 560 9 PRT Homo sapiens Residues 12-20 of the TADG-15 protein 60 Gly ProLys Asp Phe Gly Ala Gly Leu 5 61 9 PRT Homo sapiens Residues 668-676 ofthe TADG-15 protein 61 Asp Pro Thr Gln Trp Thr Ala Phe Leu 5 62 9 PRTHomo sapiens Residues 461-469 of the TADG-15 protein 62 Thr Gly Arg CysIle Arg Lys Glu Leu 5 63 9 PRT Homo sapiens Residues 59-67 of theTADG-15 protein 63 Ala Ala Val Leu Ile Gly Leu Leu Leu 5 64 9 PRT Homosapiens Residues 379-387 of the TADG-15 protein 64 Lys Val Ser Phe LysPhe Phe Tyr Leu 5 65 9 PRT Homo sapiens Residues 119-127 of the TADG-15protein 65 Lys Val Lys Asp Ala Leu Lys Leu Leu 5 66 9 PRT Homo sapiensResidues 780-788 of the TADG-15 protein 66 Leu Pro Gln Gln Ile Thr ProArg Met 5 67 9 PRT Homo sapiens Residues 67-75 of the TADG-15 protein 67Leu Val Leu Leu Gly Ile Gly Phe Leu 5 68 9 PRT Homo sapiens Residues283-291 of the TADG-15 protein 68 Ser Pro Met Glu Pro His Ala Leu Val 569 9 PRT Homo sapiens Residues 12-20 of the TADG-15 protein 69 Gly ProLys Asp Phe Gly Ala Gly Leu 5 70 9 PRT Homo sapiens Residues 257-265 ofthe TADG-15 protein 70 Ser Leu Thr Phe Arg Ser Phe Asp Leu 5 71 9 PRTHomo sapiens Residues 180-188 of the TADG-15 protein 71 Met Leu Pro ProArg Ala Arg Ser Leu 5 72 9 PRT Homo sapiens Residues 217-225 of theTADG-15 protein 72 Gly Leu His Ala Arg Gly Val Glu Leu 5 73 9 PRT Homosapiens Residues 173-181 of the TADG-15 protein 73 Met Ala Glu Glu ArgVal Val Met Leu 5 74 9 PRT Homo sapiens Residues 267-275 of the TADG-15protein 74 Ser Cys Asp Glu Arg Gly Ser Asp Leu 5 75 9 PRT Homo sapiensResidues 567-575 of the TADG-15 protein 75 Cys Thr Lys His Thr Tyr ArgCys Leu 5 76 9 PRT Homo sapiens Residues 724-732 of the TADG-15 protein76 Ser Ser Met Val Arg Pro Ile Cys Leu 5 77 9 PRT Homo sapiens Residues409-417 of the TADG-15 protein 77 Tyr Cys Gly Glu Arg Ser Gln Phe Val 578 9 PRT Homo sapiens Residues 495-503 of the TADG-15 protein 78 Thr CysLys Asn Lys Phe Cys Lys Pro 5 79 9 PRT Homo sapiens Residues 427-435 ofthe TADG-15 protein 79 Val Arg Phe His Ser Asp Gln Ser Tyr 5 80 9 PRTHomo sapiens Residues 695-703 of the TADG-15 protein 80 Lys Arg Ile IleSer His Pro Phe Phe 5 81 9 PRT Homo sapiens Residues 664-672 of theTADG-15 protein 81 Phe Arg Tyr Ser Asp Pro Thr Gln Trp 5 82 9 PRT Homosapiens Residues 220-228 of the TADG-15 protein 82 Ala Arg Gly Val GluLeu Met Arg Phe 5 83 9 PRT Homo sapiens Residues 492-500 of the TADG-15protein 83 His Gln Phe Thr Cys Lys Asn Lys Phe 5 84 9 PRT Homo sapiensResidues 53-61 of the TADG-15 protein 84 Gly Arg Trp Val Val Leu Ala AlaVal 5 85 9 PRT Homo sapiens Residues 248-256 of the TADG-15 protein 85Leu Arg Gly Asp Ala Asp Ser Val Leu 5 86 9 PRT Homo sapiens Residues572-580 of the TADG-15 protein 86 Tyr Arg Cys Leu Asn Gly Leu Cys Leu 587 9 PRT Homo sapiens Residues 692-700 of the TADG-15 protein 87 Arg ArgLeu Lys Arg Ile Ile Ser His 5 88 9 PRT Homo sapiens Residues 24-32 ofthe TADG-15 protein 88 Ser Arg His Glu Lys Val Asn Gly Leu 5 89 9 PRTHomo sapiens Residues 147-155 of the TADG-15 protein 89 Ser Glu Gly SerVal Ile Ala Tyr Tyr 5 90 9 PRT Homo sapiens Residues 715-723 of theTADG-15 protein 90 Leu Glu Leu Glu Lys Pro Ala Glu Tyr 5 91 9 PRT Homosapiens Residues 105-113 of the TADG-15 protein 91 Tyr Glu Asn Ser AsnSer Thr Glu Phe 5 92 9 PRT Homo sapiens Residues 14-22 of the TADG-15protein 92 Lys Asp Phe Gly Ala Gly Leu Lys Tyr 5 93 9 PRT Homo sapiensResidues 129-137 of the TADG-15 protein 93 Ser Gly Val Pro Phe Leu GlyPro Tyr 5 94 9 PRT Homo sapiens Residues 436-444 of the TADG-15 protein94 Thr Asp Thr Gly Phe Leu Ala Glu Tyr 5 95 9 PRT Homo sapiens Residues766-774 of the TADG-15 protein 95 Gly Glu Ile Arg Val Ile Asn Gln Thr 596 9 PRT Homo sapiens Residues 402-410 of the TADG-15 protein 96 Val GluIle Asn Gly Glu Lys Tyr Cys 5 97 9 PRT Homo sapiens Residues 482-490 ofthe TADG-15 protein 97 Asp Glu Leu Asn Cys Ser Cys Asp Ala 5 98 9 PRTHomo sapiens Residues 82-90 of the TADG-15 protein 98 Arg Asp Val ArgVal Gln Lys Val Phe 5

What is claimed is:
 1. A method of detecting in a sample TADG-15 proteinhaving an amino acid sequence of SEQ ID NO:2, comprising the steps of:(a) contacting a sample with an antibody specific for TADG-15, whereinsaid antibody is directed against the carboxy terminus of said TADG-15protein; and (b) detecting binding of said antibody to said TADG-15protein in said sample.
 2. The method of claim 1, wherein sample is abiological sample.
 3. The method of claim 2, wherein said biologicalsample is from an individual.
 4. The method of claim 3, wherein saidindividual is suspected of having cancer.